De-immunized (poly)peptide constructs

ABSTRACT

The present invention relates to a (poly)peptide construct consisting of at least two domains of at least two pluralities of domains wherein one of said domains or pluralities of domains comprises a de-immunized autoreactive antigen or (a) fragment(s) thereof specifically recognized by the Ig receptors of an autoreactive B-cells and wherein a/the further domain or plurality of domains comprises an effector molecule capable of interacting with and/or of activating NK-cells, T-cells, macrophages, monocytes and/or granulocytes. Preferably, said (poly)peptide construct consisting of at least two domains comprises a de-immunized autoreactive antigen or (a) fragment which is MOG or (a) fragment(s) thereof and a second domain comprising an effector molecule is an anti-CD3 receptor or an Fc-part of an immunoglobulin. The invention also relates to compositions comprising the compounds of the invention. Described is also the use of the afore-mentioned (poly)peptide construct and further compounds for the preparation of a pharmaceutical composition for the treatment and/or prevention of an autoimmune disease. In addition, the present invention relates to method for treating, ameliorating and/or preventing of an autoimmune disease.

The present invention relates to a (poly)peptide construct consisting ofat least two domains or at least two pluralities of domains wherein oneof said domains or pluralities of domains comprises a de-immunizedautoreactive antigen or (a) fragment(s) thereof specifically recognizedby the Ig receptors of an autoreactive B-cells and wherein a/the furtherdomain or plurality of domains comprises an effector molecule capable ofinteracting with and/or capable of activating NK-cells, T-cells,macrophages, monocytes and/or granulocytes. Preferably, said(poly)peptide construct consisting of at least two domains comprises ade-immunized autoreactive antigen or (a) fragment which is MOG or (a)fragment(s) thereof and a second domain comprising an effector moleculewhich is an anti-CD3 receptor or an Fc-part of an immunoglobulin. Theinvention also relates to compositions comprising the compounds of theinvention. Described is also the use of the afore-mentioned(poly)peptide construct and further compounds for the preparation of apharmaceutical composition for the treatment and/or prevention of anautoimmune disease. In addition, the present invention relates to methodfor treating, ameliorating and/or preventing an autoimmune disease.

Several documents are cited throughout text of this specification. Eachof the documents cited herein (including any manufacturer'sspecifications, instructions, etc.) are hereby incorporated byreference.

Autoimmunity results from the failure of the immune system in toleratingself-reactive lymphocytes, resulting in an adaptive immune responseagainst self antigens. When such immune responses are sustained, theycause lasting tissue damage and are classified as autoimmune diseases.

Autoimmune diseases are generally divided into three types: B-celldominant, T-cell dominant or combinational types. Pathogenic phenotypesof B-cell dominant autoimmune diseases are caused by autoantibodiesproduced by autoreactive B-cells, while those of the T-cell dominanttype are caused by tissue damage mediated by activated T-cells. TheseT-cells are activated by other cells presenting autoreactive peptide-MHCcomplexes on their surface. Yet, these distinctions are not perspicuous,since B-cells and T-cells cooperate with and depend on each other ineach type of autoimmune disease. Autoimmune diseases are classified ascombinatorial when both autoreactive B- and T-cells contribute directlyto the pathogenesis observed (“Immunobiology”, 4^(th) edt. (1999),Chapter 13 pp 489-536, Janeway, C. A., Travers, P., Walport, M., Capra,J. D. eds and “Harrison's Principles in Internal Medicine”,14^(th) edt,Fauci, Braunwald, Isselbacher, Wilson, Martin, Kasper, Hauser, Longo,eds).

The pathogenic effects of autoreactive B cells are caused by thesecreted autoreactive antibodies. Antibody-mediated autoimmune diseasescan be differentiated into two major groups based on theirimmunopathogenic mechanism. The first group comprises autoimmuneresponses against cell-surface or extracellular matrix antigens, whilethe second group consists of immune-complex diseases.

Examples of the first group of antibody-mediated autoimmune diseases areautoimmune hemolytic anemia, autoimmune thrombocytopenic purpura,myasthenia gravis, Goodpasture's syndrome, immunologically mediatedblistering diseases like Pemphigus vulgaris and pemphigus foliaceus, andacute rheumatic fever. Examples of the second group comprise mixedessential cryoglobulinemia, subacute bacterial endocarditis, and severalrheumatic autoimmune diseases.

Current treatment options for antibody-mediated autoimmune diseasesinclude small molecule anti-inflammatory and immuno-suppressive agents,plasmapheresis, surgical treatments and/or cytokine administration.

For example, pemphigus vulgaris and pemphigus foliaceus are usuallytreated with glucocorticoids and, in some cases, also withimmunosuppressive agents. Myasthenia gravis treatment options includeanticholinesterase medications, immunosuppressive agents, thymectomy,plasmapheresis or intravenous unspecific immunoglobulin. Multiplesclerosis treatments include interferon beta, glucocorticoids,plasmapheresis.

Yet, current therapies for autoimmune diseases/disorders are notselective, treat essentially only symptoms and/or lead to broadimmuno-suppression.

Experimental approaches to therapy include removal of the entire B cellcompartment using monoclonal antibodies against pan-B cell surfaceantigens (CD19, CD20), or even pan-leucocyte antigens (CD52). Anti-CD52monoclonal antibodies have been tested in clinical trials for theirability to ameliorate conditions of patients with multiple sclerosis.However, the indiscriminate elimination of B and T cells using anti-CD52monoclonal antibody leads to a substantial release of pro-inflammatorycytokines, contributing to a progressive phase of disability (Coles(1999) Lancet 354, 1691-1695; Coles (1999) Ann. Neurol. 46, 296-304).Thus, highly selective removal of only the autoreactive B cells isneeded to prevent the disasterous side effects. Other pan-B cellantibody based approaches include immuno-toxins (WO 96/36360). Recently,an approach combining an autoantigen with an immunotoxin has been testedin vitro. The construct was a desmoglein3 (dsg3)-toxin fusion proteinfor the treatment of experimental pemphigus vulgaris (Proby, Brit. J.Dermatol. (2000) 142, 321-330). This approach, however, lacks efficacy.In Proby (2000), a cytotoxic effect of the dsg3-PE toxin was determinedin vitro at concentrations of 50 microgram/mi. Assuming a distributionin blood and lymph fluid (about 10 l volume total) and an average weightof 70 kg, such a concentration would correspond to a dose of about 5000to 7000 micrograms/kg. In contrast, the maximal tolerated dose (MTD) inman for an anti-CD25scFv-PE toxin (LMB-2) compound was determined at 63micrograms/kg. Side effects included elevated transaminase levels andcardiomyopathy (Kreitman, (2000) J. Clin. Oncol. 18, 1622-1636). SimilarMTDs were determined for scFv-toxin LMB-1, an anti-LeY scFv-PE toxincompound (75 microgram/kg; Pai, (1996) Nat. Med. 2, 350-353). Thus, thedoses theoretically required for specific activity by dsg3-PE toxin areabout 100 times higher than the MTD determined for PE toxin compounds.It is conceivable that further harmful toxicity could result frombinding of the autoantigen-toxin construct to anti-dsg3 autoantibodies.The complex could then be bound and internalized by any kind ofFcR-bearing immune cells, killing thereby non-autoreactive immune cellsand leading to a general immunosuppression.

Myasthenia Gravis (MG) is a T cell-dependent, antibody-mediatedautoimmune disease of the neuromuscular junction, in which the nicotinicacetylcholine receptor (AchR) is the major autoantigen. Autoantibodiesto the alpha-chain of the nicotinic acetylcholine receptor present atthe neuromuscular junction block neuromuscular transmission. MG is afatal affliction; patients affected with this disease developprogressive weakness potentially leading to death. In myasthenia gravis,approaches to downregulate autoimmune reactions using intact or portionsof the acetylcholine receptor (AchR, Achr) has been the main goal ofmultiple studies in animal models. However, AChR is highly immunogenicand thus, frequent administrations of the molecule might lead to animmune response rather than tolerance induction. Therefore, shortpeptides that represent T cell epitopes of the AChR and especiallyaltered T cell epitopes with less immunogenic potential than the nativeprotein were tested in order to provide for an approach to therapy. Indetail, dominant T cell epitope peptides of the Torpedo AChR wereinjected either before immunization with the Torpedo AChR or afterpriming suppressed disease manifestations. However, at least one ofthese studies reported lack of ability of the peptides to treat anongoing disease in an animal model (Karachunski, (1999) J. Neuroimmunol.93, 108-121).

Experimental autoimmune MG (EAMG), inducible in various animal speciesby immunization with AchR or by passive transfer of anti-AchRantibodies, is a reliable model of the human disease, suitable for theinvestigation of therapeutic strategies (Fuchs, 1979, Curr. Top.Microbiol. Immunol. 85, 1-29; Drachman, 1996, Muscle Nerve 19,1239-1251).

MG is currently treated mainly by acetylcholinesterase inhibitors and bygeneralized immunosuppression. These treatments have been effective forboth MG and EAMG but are often associated with severe side effects.Ideally, the treatment should be specific and should suppressselectively the immunological reactivity that leads to the neuromusculardisorder without impairing the entire immune system. The immune responseto AchR is highly heterogeneous (Profti, 1993, Immunol. Today 14,363-368; Hawke, 1996, Immunol. Today 17, 307-311) and a wide variety ofT and B cell epitopes have been defined in MG and EAMG. Thus, the searchfor new molecules suitable for treatment of MG should deal with thisheterogeneity. Candidate molecules for antigen-specific immunotherapy ofMG should share specificities with the native antigen without beingpathogenic and should be available in sufficient amounts.

The extracellular portion of the AchR alpha-subunit is the target forthe majority of the anti-AchR antibodies in MG sera (Tzartos, 1982,Proc. Natl. Acad. Sci. USA 79, 188-192). Recombinant proteinscorresponding to this region encompass many T and B cell epitopes andcan be prepared in large amounts. They therefore represent a potentialsubstitute for the entire antigen for immunotherapy studies. Theserecombinant fragments were able to attenuate EAMG passively transferredby pathogenic monoclonal anti-AchR antibodies (Barchan, 1999, Proc NatlAcad Sci USA, 96, 8086-8091). While passive adsorption of pathogenicantibodies by recombinant autoantigen fragments has already provensuccessful in treatment of EAMG (Barchan, 1998, Eur. J. Immunol. 28,616-624), the autoreactive B cell as origin of these pathogenicantibodies is not addressed.

Approaches to treat multiple sclerosis include treatments which affectthe overall immune system, like treatment with anti-inflammatory agents,comprising azathioprine, cyclophosphamide, prednisone, corticosteroids,cyclosporin A, calcineurin, rapamycin or beta-interferon (“Harrison'sPrinciples of Internal Medicine”, 14^(th) edition, McGraw-Hillpublisher, 2415-2419; Wang, J. Immunol. 165 (2000), 548-557). Inaddition, a number of non-specific treatments are administered that mayimprove the quality of life including physical therapy andpsycho-pharmacological agents. Experimental approaches include peptideligands to block T cell epitopes (Holz, J. Immunol. 164 (2000),1103-1109; Krogsgaard, J. Exp. Med. 191 (2000), 1395-1412) and TNF alphainhibitors (Klinkert, J. Neuroimmunol. 72 (1997), 163-168).

Autoreactive B cells occur at a very low frequency. For example,autoreactive B cells circulate in the blood of individuals sufferingfrom multiple sclerosis in a frequency of about 10⁻⁶ to 10⁻⁷ of total Bcells. This low frequency has been a major impediment in the isolationof such cells from patients. Elimination of autoreactive B cells in vivocan therefore only be monitored indirectly via the determination ofautoreactive antibody titers and is further complicated by the longhalf-life of antibodies in serum.

Therefore, the technical problem underlying the present invention was todevelop and to provide for means and methods for preventing, treatingand/or ameliorating antibody-mediated autoimmune diseases/disorders.

The solution to said technical problem is achieved by the embodimentscharacterized in the claims.

Accordingly, the present invention relates to a (poly)peptide constructconsisting of at least two domains or at least two pluralities ofdomains wherein one of said domains or pluralities of domains comprisesa de-immunized, autoreactive antigen or (a) fragment(s) thereofspecifically recognized by the Ig receptors of autoreactive B-cells andwherein a/the further domain or plurality of domains comprises aneffector molecule capable of interacting with and/or capable ofactivating NK-cells, T-cells, macrophages, monocytes and/or granulocytesand/or capable of activating the complement system.

The present invention is based on the surprising finding thatcompositions as described herein are capable of selectively eliminatingautoreactive B cells and thereby removing the pathological cell causingan autoimmune disorder/disease. Furthermore, it was surprisingly foundthat the (poly)peptide constructs of the present invention are capableof inhibiting non-desired T-cell responses. In particular, theconstructs of the invention provide for (poly)peptides which are capableof a selective elimination of autoreactive B-cells and of inhibiting theinduction of T-cell activation, T-cell proliferation and/or inhibiting apathogenic event mediated by T-cells. The present invention provides fortools and methods, in particular for (poly)peptide constructs (ornucleic acid molecules encoding the same) which target (and eliminateand/or suppress) autoreactive B-cells but wherein the first domain asdescribed above does not induce T-cells/T-cell responses that maycontribute to additional problematic events in autoimmune diseases. Theinventive constructs consisting of different domains preferably mediatespecific recognition of autoreactive B-cells via at least one domain andrecruit effector cells via at least one other domain.

In accordance with the invention, it was surprisingly found that thespecific modification of T-cell regions/epitopes within an autoantigen(or fragments thereof) renders the inventive constructs/(poly)peptidesto valuable tools for treating autoimmune disorders by eliminatingautoreactive B-cells without inducing undesired T-cell responses in anindividual, preferably in a human patient.

Autoreactive B-cells are described herein above and are known to theskilled person as, e.g., illustrated in Immunobiology, Janeway andTravers, 1996 by Current Biology Ltd/Garland Publishing Inc.

B lymphocytes bear on their surface highly diverse antigen receptorswhich together are capable of recognizing a wide diversity of antigens.The antigen receptor of B lymphocytes is a membrane-bound form of theantibody that these cells will secrete when activated, see, inter alia,p. 1:6 in Immunbiology, Janeway and Travers, 1996 loc. cit. In the caseof autoreactive B cells, these antigens recognized by the antigenreceptors are self antigens. Autoimmune diseases are mediated by immuneresponses against self antigens; see p. 1-18 in Immunobiology, Janewayand Travers, 1996 loc, cit. In particular and preferably, autoreactiveB-cells are B-cells of all differentiation states, resting or activated,which carry a B-cell receptor on their cell surface and which—due tothis feature—are capable of binding to/interacting with (a) specificautoantigen(s). B-cell specific markers are also known in the art andcomprise the whole family of membrane bound Ig molecules, preferablyIgM+, IgD+, IgE+ or IgG+, preferably in combination with any of thefollowing markers B220+ (CD45R+), CD19+, CD20+, CD22+, CD21+, CD38+,CD49c+, CD72, CD79α, β+, CDw78+, MHC class II and CD43− (for resting Bcells).

Here, it was surprisingly found that constructs comprising ade-immunized autoreactive antigen or (a) fragment(s) thereof asdescribed herein may be employed for the selective elimination ofautoreactive B-cells and/or reduction of autoreactive immunoglobulinswithout inducing an undesired T-cell response, i.e. the activationand/or proliferation of (autoreactive) T-cells. This is surprising sincethe person skilled in the art would expect that the administration ofautoantigen(s) and (a) fragment(s) thereof would lead to a more profoundprevalence and/or disease state of autoimmune disorders.

The term “selective elimination” as used in accordance with the presentinvention means elimination of the above mentioned autoreactive B-cellsin vivo as well as in vitro. Said term also comprises ex vivoelimination, inter alia, by dialysis approaches. It is preferred thatsaid selective elimination does not hinder the immunological responseand/or only minimally influences the natural immunological defense. Yet,it is desired and an aim of the present invention that pathogenesismediated by T-cells and/or T-cell responses to the auto-antigen issuppressed, preferably eliminated. Preferably, said elimination ofautoreactive B-cells does not interfere with non-autoreactive B-cells.It is preferred that said elimination is caused by cytolysis, mostpreferably said cytolysis is mediated by cytotoxic cells, like,macrophages, monocytes, granulocytes, (cytolytic) T-lymphocytes, naturalkiller (NK) cells and/or lymphokine-activated killer (LAK) cells.Accordingly, triggering T-cells via the second domain, i.e. the effectordomain of the (poly)peptide of the invention is desired.

The term “at least two domains” as used herein above comprises at leasttwo domains, at least three domains, at least four domains and at leastfive domains in accordance with the invention. The term “domain” inaccordance with this invention comprises a structural and/or functionalentity of a macromolecular compound, in particular of the (poly)peptideof the invention. Said term also comprises multifunctional and/ormultiregional entities. Said domains may comprise different structuralmotifs in their secondary structure, like alpha-helices and beta-sheets.Furthermore, the term comprises at least one region of the (poly)peptideof the invention, wherein said region may also comprise severalentities. Preferably, a domain in accordance with the present inventioncomprises, at least 10, preferably at least 20, preferably at least 30,preferably at least 40, preferably at least 50, preferably at least 60,preferably at least 70, preferably at least 80, preferably at least 90,preferably at least 100, preferably at least 120, preferably at least140, preferably at least 160, preferably at least 180, preferably atleast 200, or preferably at least 220 amino acids. Accordingly, the term“domain” is related to a specific, functional tertiary structure,whereby said domain is a unit of function. Furthermore, different partsof the domains as defined herein and as comprised in the polypeptide ofthe invention may be associated with different function. The term domainalso comprises subunits of biologically active macromolecules, likeautoantigens or effector molecules as defined herein. In this context itis of note that the polypeptide of the invention may, inter alia,comprise not only one, but also several domains which comprise ade-immunized, autoreactive antigen and/or not only one, but also severaldomains comprising an effector molecule as defined herein. The term“pluralities/plurality of domains” relates, accordingly, to more thanone region of the (poly)peptide of the invention, wherein these regionsmay comprise or be structural and/or functional entities.

As employed in accordance with this invention, the term “de-immunized,autoreactive antigen or (a) fragment(s) thereof” means antigens or (a)fragment(s) therof which are capable of eliciting and/or mediating anautoimmune response. Said fragment(s) thereof is/are preferably anepitope of said antigen. The term “autreactive antigen or (a)fragment(s) thereof” as employed in the invention can be defined as aself-antigen to which autoreactive, e.g. anti-self, immunoresponses canbe raised. Preferably, said antigens and/or its fragment(s) compriseproteinaceous structures, yet, said autoreactive antigen or (a)fragment(s) may also comprise, either alone or in addition to saidproteinaceous structures, inter alia, carbohydrate moieties or lipids.Examples of autoreactive antigens which can be used in the presentinvention are, inter alia, MOG, MBP, PLP (for multiple sclerosis), Dsg3(for Pemphigus) or AchR (for myasthenia gravis). The term “autoreactiveantigen or (a) fragment(s) thereof” is not limited to antigens occurringin and/or deriving from the subjects' own body (autologous and/orendogenic antigens) but furthermore comprises foreign molecules whichare capable of eliciting an autoimmune-response by binding to and/orinteracting with molecules peculiar to one's own body (for example viahapten-carrier complexes). In addition, said term also comprisesantigens, like microbial antigens/epitopes, that share properties, e.g.amino acid sequences, with mammalian molecules, e.g. proteins, and arecapable of provoking an autoimmune-response. Examples of such antigenicmimicry are known in the art (see, inter alia, Paul, “FundamentalImmunology”, Raven Press, 1989) and comprise exogenous antigens like,Steptococcal M protein, Klebsiella nitrogenase, Measles virus P3,retroviral p30 protein or butyrophilin. It is preferred that a(poly)peptide construct of the present invention comprises a domain withat least one de-immunized, autoreactive antigen or at least one fragmentthereof. However, it is also envisaged that said (poly)peptide constructcomprises a domain comprising more than one de-immunized, autoreactiveantigens and/or fragments and/or epitopes thereof. Said domaincomprising said de-immunized, autoreactive antigen or (a) fragmentthereof may therefore comprise several autoantigens and/or fragment(s)thereof. In a preferred embodiment said domain comprises at least one,more preferred at least two, more preferred at least three, morepreferred at least four and more preferred at least five de-immunized,autoreactive antigen(s) or (a) fragment(s).

The term “de-immunized, autoreactive antigen or (a) fragment(s) thereof”relates to autoreactive antigen(s) or (a) fragment(s) thereof as definedherein above, wherein the term “de-immunized” relates to the specificremoval and/or modification of T-cell epitopes/domains from saidautoreactive antigen/autoreactive fragment.

The term “de-immunized” is well known in the art and, inter alia,employed for the removal of T-cell epitopes from a (therapeutic)antibody; see WO 98/52976 or WO 00/34317.

De-immunization involves, in accordance with the invention, theidentification, modification and/or removal of T-cell epitopes,preferably helper T-cell epitopes. In this context, the term T-cellepitope relates to T-cell epitopes comprising small peptides which arerecognized by T-cells in the context of MHC class II molecules. Thisrecognition may be accompanied by activation of the T-cell and secretionof pro-inflammatory cytokines.

This type of T-cell activation is not related to the antigen-independenteffector cell activation induced by the effector domain of the inventiveconstructs. These constructs mediate the recruitment of effector cells(p.e. T-cells) inducing a deletion of the specific B-cells.

Methods for the identification of such T-cell epitopes are known in theart (see, inter alia, WO 98/52976, WO 00/34317) and are, inter alia,illustrated in the appended examples. The methods comprise, e.g. peptidethreading, peptide-MHC binding, human T-cell assays analysis of cytokineexpression patterns, ELISPOT assays, class II tetramer epitope mapping,search of MHC-binding motif databases and the additionalremoval/modification of T-cell epitopes.

Peptide Threading is a technique based on the analysis of peptides thatbind to MHC class II molecules (major histocompatibility antigen, alsoknown as leukocyte antigen or HLA). By using a combination of known HLAthree dimensional structures and homology modelling, the structures ofmany human MHC alleles have been predicted. Peptides are known to bindto MHC class II via a cleft which has pockets radiating from it toaccommodate the amino acid side chains. All overlapping peptidescovering the whole antibody or protein sequence of choice are assessedfor binding to MHC II in silico and a binding score is calculated.

In contrast to the in silico method of Peptide Threading, the in vitromethod of peptide-MHC binding uses a collection of human cell linescarrying a repertoire of different MHC class II alleles. Typically,synthetic peptides from antibody and protein sequences are tested fordisplacement of control biotinylated peptides.

Following cell lysis, MHC class II molecules are immunoprecipitated andtested for peptide binding using avidin-enzyme conjugates. Peptide-MHCbinding data shows an excellent correlation with Peptide Threading andprovides concise data for a wide range of MHC allotypes.

Unlike Peptide Threading and peptide-MHC binding which measure events onthe antigen-presenting cell, human T cell assays measure the T cellresponse to peptides presented in conjunction with MHC class IImolecules. Peptides or proteins are mixed with human antigen presentingcells and T cells are added. T cell proliferation in response to thespecific antigens is then assessed by tritiated thymidine uptake orcytokine measurement. Determination of cytokine pattern may be performedon protein or mRNA level. Human T cell assays are used to identifypeptide-MHC class II complexes which can trigger T cell responses.

Having identified T cell epitopes by application of the above-recitedtechnologies, these can be eliminated, substituted and/or modified fromthe autoantigen or from (a) fragment(s) thereof, usually by single aminoacid substitutions within the MHC class II binding peptide; asillustrated in the appended examples and further described herein below.While such substitutions will eliminate or greatly reduce binding to MHCclass II, an alternative strategy involves altering the MHC bindingpeptide to a sequence which retains its ability to bind MHC class II butfails to trigger T cell activation and/or proliferation.

As illustrated in the examples, removal of immunodominant epitope of,for example, MOG may be carried out as follows:

Following identification of the immunodominant epitope of the MOGextracellular domain, the epitope comprising a given sequence of aminoacids can be modified to remove the potential of said peptide tostimulate T cells. Said modifications may comprise substitutions of oneor more amino acids of the epitope to any given amino acid, preferablyto alanine. Modifications may also comprise deletion of one or moreamino acids of the epitope. Such modifications can be introduced intothe peptide by standard chemical peptide synthesis. Modificationscomprising substitutions and/or deletions of one or more amino acids canbe incorporated into the MOG-Fc protein by molecular biology procedures.

The construct may be tested by methods mentioned above. For oneillustrative example, namely an de-immunized eMOG-Fc construct, thefollowing assays may be carried out. It is of note, that such assays mayalso be employed for further inventive constructs.

a) T-cell stimulation assay with a newly generated MOG-reactive T-cellline, wherein proliferation in response to eMOG-Fc (deimmunized MOGfusion protein) stimulation is measured:

MOG-reactive T cell line was generated by standard protocol. Briefly,SJL/UJ mice were immunized with recombinant MOG protein (rMOG) inComplete Freund's Adjuvans (CFA). Following immunization, spleen anddraining lymph nodes were prepared. Single-cell cultures wereestablished. Periodically, cells were re-stimulated with irradiatedantigen-presenting cells (APC) loaded with rMOG, thereby selecting forMOG-reactive T cells.

The newly established MOG-reactive T-cell line was used in a T-cellproliferation assay: APCs were loaded with human IgG1, rMOG (recombinantMOG) and eMOG-Fc proteins. The proliferative response of rMOG-reactiveT-cell line was tested in standard 3-H thymidine incorporation assay.Proliferation to negative IgG1 control was comparable to theproliferative response to eMOG-Fc protein.

b) Determination of cytokine pattems of primary murine T cells, whereinproliferation in response to eMOG-Fc; or induction of a non-Th1 cytokineprofile be ensured:

SJL/J mice were immunized with human IgG1, rMOG or eMOG-Fc in completeFreund's Adjuvants (CFA). Following immunization, spleen and draininglymph nodes were prepared and blood was taken. Single-cell suspensionswere prepared, and the functional phenotype of cells was analyzed byFACS. Additionally, single-cell cultures were established. The cytokineprofile in the supernatant was detected by ELISA (BD OptElA ELISA Set).Interestingly, mice immunized with eMOG-Fc displayed no detectablecytokine secretion or, if detectable, a predominantly Th2-mediatedcytokine pattern (IL10 high, IL4 high, IFNg negative). This is incontrast to the group of mice immunized with rMOG, which presented witha strong Th1 cytokine profile (IFNγ high, TNFα high).

c) Immunization of mice with eMOG-Fc in CFA, wherein absence of diseaseinduction is measured:

6-8 week-old Female SJUJ mice were immunized in the hind foodpads with100 μg protein in 100 μl Complete Freund's Adjuvants (CFA) at 1:1 v/v.Following immunization, animals were scored daily on induction ofExperimental Autoimmune Encephalomyelitis (EAE) on the standard EAEscale of 0 (healthy) to 5 (moribound or dead). Weight was recordeddaily. Animals were devided into the following groups (n=5) immunizedwith: 1) rMOG, 2) EpCAM, 3) eMOG-Fc.

Animals immunized with control human IgG1 against EpCAM showed noclinical symptoms. In contrast, immunization with rMOG led to a rapidlyprogressive disease resulting in an average EAE clinical score greaterthan 3. Animals immunized with eMOG-Fc showed no induction of clinicalEAE manifestation. Since EAE requires CNS-specific T cells to mediateimmunopathology, the absence of disease in the eMOG-Fc immunizedanimals, but not the rMOG immunized animals proves that the concept of“de-immunizing” the MOG extracellular domain works in clinical MS-likedisease models.

It is of note that, in accordance with this invention, the term“de-immunized” relates to the first domain of the inventive(poly)peptide construct as described herein above, i.e. to theautoreactive antigen or (a) fragment(s) thereof. Accordingly, the(poly)peptide construct of the present invention comprises anautoreactive antigen or (a) fragment(s) thereof, wherein immuno-dominantT-cell epitopes comprised in said antigen have been removed, substitutedand/or modified. As pointed out above, a particular useful way fordetermining/identifying immuno-dominant T-cell epitopes comprisestechniques also illustrated in the examples. It is also envisaged thatcritical T-cell receptor contact residues within a given epitope may beidentified by single amino acid point mutations, e.g. to Alanine(Alanine-scan). In accordance with this invention, MHC class II motifsmay be identified and de-immunized through predictive algorithms(peptide threading) or a combination of computer-assisted algorithms andclass II tetramer epitope mapping (Kwok et al, Trends in Immunology 22:583 (2001)). This approach aolishes class II binding and subsequentT-cell receptor activation.

The term “Ig receptor” means the cell-surface immunoglobulin (Ig) foundon B cells, also known as the B-cell receptor (BCR).

The term “effector molecule capable of interacting with and/oractivating NK-cells, T-cells, macrophages, monocytes and/orgranulocytes” relates, in accordance with this invention, to moleculescapable of engaging, inter alia, lymphocytes and/or FcγR positive cellsin effector mechanisms, like cell lysis and/or phagocytosis. Saidlymphocytes and/or FcγR positive cells comprise the above mentionedNK-cells, macrophages, monophages, monocytes and/or granulocytes, aswell as lymphokine-activated killer cells, neutrophiles or eosinophils.In accordance with this invention, the term “effector molecule capableof interacting with and/or activating NK-cells, T-cells, macrophages,monocytes and/or granulocytes” also relates to functional fragments ofsaid effector molecules, i.e. to fragments of said molecules, which arecapable of interacting with and/or capable of activating the cellsdefined herein above. As discussed herein above, the term “domain” asused in accordance with the invention is not limited to a singlestructural and/or functional motif or entity in the polypeptide of theinvention but may also comprise several units of function, i.e. effectorfunction or the function of a de-immunized, autoreactive antigen or (a)fragment(s) thereof.

In accordance with the present invention, the term “effector moleculecapable of activating the complement system” relates to effectormolecules which are capable of activating the classical as well as thealternative complement pathway. Furthermore, said term relates toeffector molecules capable of activating any other form of complementmediated lysis.

Useful “effector molecules” in accordance with the present inventionare, inter alia, disclosed herein and exemplified in the appendedexamples.

In a preferred embodiment, the present invention relates to a(poly)peptide construct which is a fusion (poly)peptide or a mosaic(poly)peptide. Said fusion (poly)peptide may comprise merely the domainsof the (poly)peptide construct as described herein above as well asseveral (a) functional fragment(s) thereof. However, it is alsoenvisaged that said fusion poly)peptide comprises further domains and/orfunctional streches. Therefore, said fusion (poly)peptide can compriseat least one further domain, said domain being linked by covalent ornon-covalent bonds. The linkage (as well as the construction of the(poly)peptide constructs comprised in the composition of the presentinvention), can be based on genetic fusion according to the methodsknown in the art (Sambrook et al., loc. cit., Ausubel, “CurrentProtocols in Molecular Biology”, Green Publishing Associates and WileyInterscience, N.Y. (1989)) or can be performed by, e.g., chemicalcross-linking as described in, e.g., WO 94/04686. The additional domainpresent in the fusion (poly)peptide may preferably be linked by aflexible linker, advantageously a (poly)peptide linker, wherein said(poly)peptide linker preferably comprises plural, hydrophilic,peptide-bonded amino acids of a length sufficient to span the distancebetween the C-terminal end of said further domain and the N-terminal endof the peptide, (poly)peptide or antibody or vice versa. Said linkermay, inter alia, be a Glycine, a Serine and/or a Glycine/Serine linker.Additional linkers comprise oligomerization domains. Oligomerizationdomains facilitate the combination of two or several autoantigens orfragments thereof in one functional molecule. Non-limiting examples ofoligomerization domains comprise leucine zippers (like jun-fos, GCN4,E/EBP; Kostelny, J. Immunol. 148 (1992), 1547-1553; Zeng, Proc. Natl.Acad. Sci. USA 94 (1997), 3673-3678, Williams, Genes Dev. 5 (1991),1553-1563;Suter, “Phage Display of Peptides and Proteins”, Chapter 11,(1996), Academic Press), antibody-derived oligomerization domains, likeconstant domains CH1 and CL (Mueller, FEBS Letters 422 (1998), 259-264)and/or tetramerization domains like GCN4-LI (Zerangue, Proc. Natl. Acad.Sci. USA 97 (2000), 3591-3595).

Furthermore, the (poly) peptide construct as described herein maycomprise further domains, inter alia, domains which provide forpurification means, like, e.g. histidine stretches.

It is also envisaged that the (poly)peptide construct as describedherein comprises (a) further domain(s) which may function asimmunomodulators. Said immunomodulators comprise, but are not limited tocytokines, lymphokines, T cell co-stimulatory ligands, etc. Preferably,said domains may comprise regions of IL-4 molecule.

Adequate activation resulting in priming of naive T-cells is critical toprimary immunoresponses and depends on two signals derived fromprofessional APCs (antigen-presenting cells) like dendritic cells. Thefirst signal is antigen-specific and normally mediated by stimulation ofthe clonotypic T-cell antigen receptor that is induced by processedantigen presented in the context of MHC class-I or MHC class-IImolecules. However, this primary stimulus is insufficient to inducepriming responses of naive T-cells, and the second signal is requiredwhich is provided by an interaction of specific T-cell surface moleculesbinding to co-stimulatory ligand molecules on antigen presenting cells,further supporting the proliferation of primed T-cells. The term “T-cellco-stimulatory ligand” therefore denotes in the light of the presentinvention molecules, which are able to support priming of naive T-cellsin combination with the primary stimulus and include, but are notlimited to, members of the B7 family of proteins, including B7-1 (CD80)and B7-2 (CD86).

In the light of the present invention, proteinaceous compounds providingthe primary activation signal for T-cells can comprise, but are notlimited to, anti-CD3-scFv fragments, anti-T-cell receptor scFv fragmentsor superantigens. Superantigens directly bind to certain subfamilies ofT-cell receptor variable regions in an MHC-independent manner thusmediating the primary T-cell activation signal.

Furthermore, the invention also relates to the effector molecule asdefined herein, wherein the T-cell co-stimulatory ligand is a cellsurface molecule or a fragment thereof expressed on antigen-presentingcells (APC).

Additionally, the effector molecule as defined herein, binding to anAPC, may be a T-cell co-stimulatory factor like B7-1 (CD80) or B7-2(CD86), or adhesion proteins like LFA-3 (CD58), ICAM-1 (CD54), ICAM-2 orICAM-3 or like the CD137-ligand.

The effector molecule defined herein above may have receptor or ligandfunction, and may be an immuno-modulating effector molecule or afragment thereof. An immuno-modulating effector molecule positivelyand/or negatively influences the humoral and/or cellular immune system,particulary its cellular and/or non-cellular components, its functions,and/or its interactions with other physiological systems. Saidimmuno-modulating effector molecule may be selected from the groupconsisting of cytokines, chemokines, macrophage migration inhibitoryfactor (MIF; as described, inter alia, in Bernhagen (1998), Mol Med76(3-4); 151-61 or Metz (1997), Adv Immunol 66,197-223) , T-cellreceptors and soluble MHC molecules. Such immuno-modulating effectormolecules are well known in the art and are described, inter alia, inPaul, “Fundamental immunology”, Raven Press, New York (1989). Inparticular, known cytokines and chemokines are described in Meager, “TheMolecular Biology of Cytokines” (1998), John Wiley & Sons, Ltd.,Chichester, West Sussex, England; (Bacon (1998). Cytokine Growth FactorRev 9(2):167-73; Oppenheim (1997). Clin Cancer Res 12, 2682-6; Taub,(1994) Ther. Immunol. 1(4),229-46 or Michiel, (1992). Semin Cancer Biol3(1),3-15).

Particularly preferred are cytokines which are selected from the groupconsisting of interleukin(s), interferon(s), TNF(s) and VEGF (Veikkola(1999) Semin Cancer Biol 9(3), 211-20), wherein said interleukin(s)comprise, but are not limited to IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-12, IL-13, IL-14, IL-15,IL-16, IL-17, IL-18 and IL-21, wherein interferon(s) comprise IFN-γ aswell as IFN-β and IFN-α and wherein TNF(s) comprise members of thelymphotoxin superfamily like TNF-α and TNF-β (Gruss (1996) Int J ClinLab Res 26(3),143-59). Other suitable cytokines are well known in theart and comprise, inter alia, GM-CSF, G-CSF, M-CSF. In a particularpreferred embodiment, said immuno-modulating effector molecule is achemokine and is selected from the group consisting of IL-8, Eotaxin,GROα, GROβ, GROγ, IP-10, MCP-1, MCP-2, MCP-3, MCP4, MIG, MIP-1α, MIP-1β,NAP-2, RANTES, 1309, Lymphotactin, SDF-1 and C5a.

Further effector molecules may be selected from the group ofneuroprotective proteins, such as the neurotrophic growth factors.Examples of such molecules are the neurotrophins nerve growth factor(NGF), brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3),and/or the growth factors IGF-1 and bFGF, and/or the respectivereceptors of the aforementioned molecules (Connor B., Brain Res. BrainRes. Rev. 27 (1998), 1-39; Lewin, Annual Review of Neuroscience 19(1996), 289-317; Tessarollo, Cytokine Growth Factor Rev. 9 (1998),125-137; Snider, Cell 77 (1994), 627-638; Garcia-Estrada, Brain Res. 592(1992), 3443-347; Lindsay, Trends in Neurosciences 17 (1994), 182-190;Minichiello, Genes Development 10 (1996), 2849-2858). The therapeuticpotential of NGF treatment in autoimmune encephalomyelitis has beenshown by Villoslada et al. in the marmoset EAE model (J. Exp. Med. 191(2000),1799-1806).

Within the scope of the present invention are furthermore effectormolecule comprising, inter alia, different immunomodulating effectormolecules. Particularly preferred are effector molecules are cytokines,like IL-2 and GM-CSF.

The (poly)peptide constructs described herein or comprised in thecomposition of the present invention may be constructs which comprisedomains originating from one species, preferably from mammals, morepreferably from human. However, chimeric and/or humanized constructs arealso envisaged and within the scope of the present invention.

In a particularly preferred embodiment, the (poly)peptide of theinvention comprises a construct which is a cross-linked (poly)peptideconstruct. As mentioned herein above, said cross-linking may be based onmethods known in the art which comprise recombinant as well asbiochemical methods.

The present invention relates in a further embodiment to a (poly)peptideas described herein above, wherein said de-immunized autoreactiveantigen or (a) fragment(s) thereof is selected from the group consistingof intracellular matrix proteins, extracellular matrix proteins,complement factors, nuclear antigens, cell surface receptors, nuclearreceptors, lipoproteins, soluble factors, membrane proteins, heat shockproteins, proteins with sequence similarity to microbial antigens,dietary components and proteins of intercellular structures.

In a more preferred embodiment, said intracellular matrix protein isselected from the group consisting of keratin, filaggrin andantiperinuclear factor 7. So far, rheumatoid factor (RF), an IgMautoantibody directed against the Fc region of IgG2a, is still the onlywell-established serological disease marker for rheumatoid arthritis(Tighe (1997) in “Textbook of Rheumatology” W. B. Saunders Company,Philadelphia, Pa. 241-249.). However, rheumatoid factor is not specificfor rheumatoid arthritis (RA) and is often negative in the early stagesof the disease when a definite diagnosis is not always possible. In thepast few years several new autoantibodies have been described which maybe more specific for RA than rheumatoid factor. Among these are anti-A2/RA33 antibodies (Hassfeld (1989) Arthritis Rheum. 32: 1515-1520;Hassfeld (1993) Br. J. Rheumatol. 32: 199-203), antikeratin antibodiesand the antiperinuclear factor 7 (Youinou (1995) Int. Arch. AllergyImmunol. 107: 508-518; Sebbag (1995) J. Clin. Invest. 95: 2672-2679),and anti-Sa antibodies (Dèspres, N., G. Boire, F. J. Lopez-Longo, and H.A. Menard (1994) J. Rheumatol. 21: 1027-1033). Anti-A2/RA33autoantibodies are directed to the RNA binding region of the A2 proteinof the heterogeneous nuclear ribonucleoprotein complex (Skriner, J.Clin. Invest. 100 (1997), 127-135). Antibodies to the Sa antigen(streptococcal antigen) cross-react in an example of molecular mimicrywith a poorly soluble human protein that is present in normal tissuesand that is distinct from all previously described RA-associatedautoimmune systems (Depres, J. Rheumatol. 21 (1994), 1027-1033).

Furthermore, the cytokeratin filament-aggregating protein filaggrin isthe target of the so-called “antikeratin antibodies,” autoantibodiesspecific for rheumatoid arthritis (Simon (1993) J. Clin. Invest.92,1387-93).

In yet another embodiment, the present invention relates to a(poly)peptide as described herein above, wherein said extracellularmatrix protein is collagen. It is particularly preferred that saidcollagen is collagen type IV or collagen XVII. In this context, theautoreactive antigen or (a) fragment thereof may also be thenon-collagenous domain of collagen.

Goodpasture's syndrome results from antibodies directed against collagentype IV, in particular the non-collagenous domain of the basementmembrane collagen type IV (Butkowski et al. (1987) J. Biol. Chem. 262,7874-7877; Saus et al. (1988) J.Biol.Chem. 263, 13374-13380). Theclinical manifestations of Goodpasture's syndrome are glomerulonephritisand pulmonary hemorrhage (Wilson, C., and Dixon, F. (1986) in the kidney(Berner, B., and Rector, F. eds) 3^(rd) Ed., pp 800-889, W.B. SaundersCo., Philadelphia).

Serum levels of autoantibodies to hemidesmosomal collagen XVII/BP180were reported to correlate with disease activity in patients withbullous pemphigoid (see herein below). Autoantibodies against the 180kDa full-length, transmembrane protein of collagen XVII and a recentlyidentified 120 kDa soluble fragment that corresponds to its collagenousectodomain were detected in patients with pemphigoid and linear IgAdermatosis (Roh, B. J. Dermatol., 2000, 143, 104-111).

In a further embodiment of the present invention, the above mentionedcomplement factor is C5. It has been shown that the complement factor C5is a potent autoantigen in rheumatoid arthritis (Volkman, J. Immunol.158 (1997), 693-706; Grant, Cell Immunol. 167 (1996), 230-240).

Furthermore, in a more preferred embodiment, the present inventionrelates to the above described (poly)peptide wherein said nuclearantigen is selected from the group consisting of DNA, histones, snRNPs,topoisomerase I, ro (SS-A-Ro), Ia (SS-B-La), ScI-70, centromer protein(CENP) AL Sm proteins, tRNA synthetase and Ku antigen.

A characteristic feature of rheumatic autoimmune diseases such asSystemic lupus erythematosus (SLE), progressive systemic sclerosis,polymyositis, mixed connective tissue disease (MCTD), or RA is theoccurrence of autoantibodies to intracellular antigens (von Muehlen(1995) Semin. Erthritis Rhem. 24, 323-358; Peng (1997) in “Textbook ofRheumatology.” W.B. Saunders Company, Philadelphia, Pa. 250-266). Forreasons which are not yet fully understood, these autoantibodies areoften directed to components of large ribonucleoprotein (RNP) structuressuch as the ribosome or the spliceosome (van Venrooij (1995) Curr. Opin.Immunol. 7: 819-824). Some of these autoantibodies specifically occur inonly one disease, which makes them very useful for diagnosis andtreatment. Thus, autoantibodies to double-stranded DNA or to the Smantigen (Brahms, JBC 275 (2000), 17122) are highly specific for SLE,autoantibodies to topoisomerase (anti-Scl70) (Mukai, J. Rheumatol. 20(1993), 1594-1497, van Venrooij, Curr. Opin. Immunol. 7 (1995), 819-824)are exclusively detected in patients with progressive systemicsclerosis, and autoantibodies to tRNA synthetases (e.g., anti-Jol)(Marguerie, Q. J. Med. 77 (1990), 1019-1038) occur only in patients withpoly- or dermatomyositis.

Systemic lupus erythematosus (SLE) is an idiopathic autoimmune diseasein which self-reactive autoantibodies (Cabral (1997); Curr. Opin.Rheumatol. 9, 387-392) cause disease either by directly binding toself-antigens or following the deposition of antibody-antigen immunecomplexes in blood vessels leading to vasculitis, glomerulonephritis andarthritic tissue damage (Rothfield (1985) in “Arthritis and AlliedConditions”, Lea & Febiger, Philadelphia, pp. 911-935). The estimatedprevalence of SLE in the U.S. is 45/100,000, with the peak incidence inwomen of ages 20-40 (Hochberg (1997) in “Dubois' Lupus Erythematosus”,Williams & Wilkins, Baltimore, pp. 49-65). Proteins cleaved byinterleukin-1 beta converting enzyme family proteases during apoptosis,as well as the Sm proteins B/B′, D1, and D3 of the spliceosome, arecommon targets for autoantibody production in patients with systemiclupus erythematosus (SLE) (Brahms (2000) JBC 275, 17122 ff.). Inaddition, immune responses to SS-A 52-kDa and 60-kDa proteins and toSS-B 50-kDa protein have been shown in mothers of infants with neonatallupus erythematosus (Yukiko (2000) Br. J. Dermatol. 142, 908-912).

Photosensitivity of lupus erythematosus was correlated with theexpression of SS-A/Ro and SS-B/La antigens in skin biopsy specimens ofpatients (loannides (2000) Arch Dermatol 136, 340-346). Wang et al. (J.Clin. Invest. (1999) 104, 1265-1275) have identified a novel 75-kDaphosphoprotein associated with SS-A/Ro and distinct human autoantibodiesdirected against it. Circulating anticentromere CENP-A and CENP-Bantibodies were identified in patients with diffuse and limited systemicsclerosis, systemic lupus erythematosus, and rheumatoid arthritis (Russo(2000) J Rheumatol. 27, 142-148). Ku is a heterodimeric protein composedof approximately 70- and approximately 80-kDa subunits (Ku70 and Ku80)originally identified as an autoantigen recognized by the sera ofpatients with autoimmune diseases (Tuteja (2000) Crit Rev Biochem MolBiol 35, 1-33).

The present invention relates furthermore to a (poly)peptide asdescribed herein wherein said de-immunized, autoreactive antigen or (a)fragment thereof is a cell surface receptor and wherein said cellsurface receptor is selected from the group consisting of TSH-receptor,Ach-receptor, asialo-glycoprotein receptor and platelet integrinGpIIb:IIIa.

Grave's disease is an autoimmune condition characterized typically byhyperthyroidism, thyroid hyperplasia, and additional signs ofophthalmopathy, pretibial myxedema, or acropachy. The pathophysiologicalmechanisms responsible for thyrotoxicosis and thyroid hyperplasia areattributed to autoantibodies directed against the thyrotropin receptor(TSHr). Said antibodies activate the TSH receptor, which results incAMP-dependent stimulation of thyrocyte function and growth (McKenzien(1995) in “Endocrinology”. “;. W. B. Saunders Co. Philadelphia, Pa.; pp676-711).

Autoantibodies directed against the acetylcholine receptor(Ach-receptor, AchR) are involved in Myasthenia gravis, an autoimmunedisease which leads to a reduction of the number of (Ach-R) at themuscular motor endplate (see, inter alia, Heitmiller (1999) Semin.Thorac Cardiovasc. Surg., 11, 41-46 or Atassi (1997) Crit. Rev.Immunol.; 17, 481-495). In addition, autoantibodies directed against thehuman asialo glycoprotein receptor are described in autoimmune hepatitis(Bojic (1997) Med. Pregl. 50, 363-8).

Furthermore, autoantibodies against platelet integrin GpIIb:IIIa are thecause for autoimmune thrombocytopenic purpurea resulting in abnormalbleeding (Beardsley and Ertem (1998) Transfus. Sci. 19, 237-244).Myasthenia gravis is caused by autoantibodies against the nicotinicacetylcholine receptors (AchR) leading to the downregulation ofreceptors and complement dependent lysis of the neuromuscular junction.The consequences are defects in neuromuscular transmission, culminatingin weakness and fatigue of skeletal muscles in MG patients (Fambrough(1973) Science 182, 293-295; Kao (1977) Science, 196, 527-529; Heinemann(1977) Proc. NatI Acad. Sci. USA 7, 3090-3094).

In an even more preferred embodiment, the present invention provides fora (poly)peptide of the invention wherein the soluble factor mentionedherein above is selected from the group consisting of I-antigen,Rh-blood group factor, 21-hydrolase enzyme, glutamic acid decarboxylase(GAD), insulin, (ICA) 512, ICAP-69, (tissue) transglutaminase (tTG),transaldolase, S100beta, oxidized low-density lipoprotein (ox-LDL),crystallin, CNPase, proteinase 3 and type I antigen.

Autoimmune hemolytic anemia is caused by antibodies against Rh bloodgroup antigens and type I antigens, destroying red blood cells andresulting in anemia (Leddy (1993); J. Clin Invest. 91,1672-1680; Leddy(1994); 84, 650-656). Celiac disease, also refered to gluten sensitiveenteropathy is characterized by IgA autoantibodies against anti-tissuetransglutaminase and antiendomysial antibodies (EMA) (Lock, R. J. (1999)J Clin Pathol 1999 Apr;52(4):274-277; Rose, C. (1999) J Am Acad Dermatol41, 957-961; Vitoria, J. C. (1999) J Pediatr Gastroenterol Nutr 29,571-574; Schuppan, D. (2000) Gastroenterology 119, 234-242).

Auto-antibodies to oxidized low-density lipoprotein (ox-LDL) are thoughtto play a pivotal role in the pathogenesis of atherosclerosis and canserve as a marker of coronary artery disease in patients with familialhypercholesterolaemia (Paiker (2000) Ann Clin Biochem 37, 174-178).

Insulin-dependent diabetes mellitus (IDDM) is traditionally classifiedas a T-cell dependent autoimmune disease. However autoreactiveantibodies are present in patients with IDDM. In particular, maternalautoantibodies may contribute to the development of juvenile IDDM.Furthermore, contributions of the humoral response to the onset ofdisease and disease progression of IDDM was shown (Bonifacio (2000)Diabetes 49, 202-208; Coleman (2000) Diabetologia 43, 203-209; Rulli, M.(1999) Autoimmunity 31, 187-193). Also a connection betweenautoantibodies to diabetes mellitus and celiac disease was shown(Galli-Tsinopoulou A (1999) Horm Res 52, 119-124). A major target ofautoimmunity in preclinical type 1 diabetes is glutamic aciddecarboxylase, GAD, specific examples are GAD65 and GAD67 (Bonifacio(2000) Diabetes 49, 202-208). Islet cell autoantigen (ICA) 512 is anautoantigen of insulin-dependent diabetes mellitus (IDDM) which ishomologous to receptor-type protein tyrosine phosphatases (++PTPases).ICA 512 is an intrinsic membrane protein of secretory granules expressedin insulin-producing pancreatic beta-cells as well as in virtually allother peptide-secreting endocrine cells and neurons containingneurosecretory granules (Solimena (1996) EMBO J 15, 2102-2014). Otherautoantigens in IDDM are ICAp69, (Karges (1996) Diabetes 45, 513-521).Anti-insulin antibodies were shown to be linked to the onset of diabetes(Yu (2000) Proc Natl Acad Sci USA 97, 1701-1706)

Granulomatosis also known Morbus Wegener (Hewis, Curr. Opin. Rheumatol.12 (2000), 3-10) is caused by autoantibodies against proteinase 3, aconstituent of neutrophil azurophilic granules.

In addition, the present invention provides for an inventive(poly)peptide as described herein above, wherein said heat shock proteinis selected from the group consisting of alpha B-crystallin, Hsp27,HSP70 and HSP60. Alpha B-crystallin and Hsp27 have been implied inmultiple sclerosis (Agius, Acta Neurol. Scand. 100 (1999), 139-147).Furthermore, antibodies to mycobacterial heat shock proteins bind tohuman myelin and to oligodendrocytes recognizing human autoantigens,including HSP70 and myelin protein CNP (Salvetti, J. Neuroimmunol. 64(1996), 143-153; Salvetti, J. Autoimmun. 5 (1992), 691-702; Birnbaum,Ann. N.Y. Acad. Sci. 835 (1997), 157-167; Jones, Immunol. Today 14(1993), 115-118). Antibodies against Escherichia coli and chlamydialHsp60 were shown to crossreact with human HSP60 (Mayr, Circulation 99(1999),1560-1566). Retinal autoantigens include Hsc70 (Ohguro, Invest.Ophthalmol. Vis. Sci. 40 (1999), 82-89).

As will be discussed in detail herein below, several proteins have beenidentified that can act as autoantigens in multiple sclerosis, includingthe soluble proteins butyrophilin, CNPase, Transaldolase, S100β,B-crystallin and other heat shock proteins (see, Schmidt (1999) MultipleSclerosis 5, 147-160; Bajramovic (2000) J Neuroimmunol 106,14-22;Steffert (2000) J. Immunol. 165:2859-2865).

The present invention relates, in a more preferred embodiment, to a(poly)peptide as described herein above, wherein said membrane proteinis selected from the group consisting of PLP, MAG, MBP, MOG,Golgi-proteins, cytochrome P450 (CYPs), UDP-glucuronosyltransferase(UGTs), pemphaxin (Nguyen, J. Biol. Chem., 2000, 275, 29466-29476) andLAD285 (Palmer, Br.J. Immunol., 2001, 145, 816-120 and Collier,Dermatology, 1994, 189, Suppl. 1,105-107).

Multiple sclerosis (MS) is a chronic inflammatory disease of the centralnervous system (CNS) of autoimmune origin, characterized by focaldemyelination, loss of oligodendrocytes, and astrocytic scar formationin advanced stages of the disease. Histopathologically, acuteinflammatory lesions are characterized by infiltrating lymphocytes andmacrophages scattered throughout the periventricular white matter,spinal cord, brainstem and optic nerves. In the later stages of thedisease, vascular infiltrates are less prominent, and loss of myelin andoligodendrocytes predominates. While MS has been widely classified to bea majorly T-cell mediated disease, it is now recognized that MS has botha T-cell and a B-cell component (Ewing (1998) Immunology and CellBiology 76, 47-54; Weckerle (1999) Nature Medicine 5, 153-154; Genain(1999) Nature Medicine, 5, 170-175; Schmidt (1999) loc.cit; Lindert(1999) Brain 122, 2089-2099). Eventually, myelin breakdown as thehallmark of the disease is brought about by the combined effects ofautoantibodies against myelin proteins, complement activation, cytotoxiccells and cytokine-induced toxicity.

As mentioned herein above, several soluble proteins have been identifiedas being involved as autoantigens in multiple sclerosis, including theabove discussed soluble proteins like, CNPase, transaldolase, S100β orB-crystallin and/or other heat shock proteins. However, further proteinsplay major roles in MS. These proteins comprise myelin basic protein(MBP), myelin oligodendrocyte glycoprotein (MOG), PLP and MAG (Schmidt(1999) loc.cit.). While it was long thought that the major myelincomponent MBP was the major target antigen in MS, studies of theexperimental animal model of MS, experimental autoimmuneencephalomyelitis (EAE), have suggested that other candidateautoantigens may be more relevant to the disease. Indeed, the MOGprotein, a minor constituent of myelin sheaths (0.05% of total myelin)and exclusively expressed as a cell-surface protein on their outermostsurface layer, has been shown to be the only single protein able toinduce Chronic relapsing EAE (CREAE) in the Lewis Rat. CREAE is thoughtto be the most appropriate animal model for MS. MOG is a membraneglycoprotein found predominantly in the outer-most lamella of the myelinsheaths that wrap and insulate axons in the CNS. It is a minor proteincomponent of myelin representing just 0.05% of the total proteincontent, in contrast to other myelin proteins with encephalitogenicpotential such as proteolipid protein (PLP) making up 20% of myelinprotein. Nonetheless, immunization with MOG induces severe chronic EAEcharacteristically accompanied by strong inflammatory and de-myelinatinglesions of the CNS (Iglesias (2001) Glia, 36:220-234). It wasdemonstrated that MOG-induced EAE comprises an important pathogenicantibody component (Schluesener, J. Immunol. 139, (1987), 4016-4021 andLitzenburger, J. Exp. Med. 188, (1998), 169-180). Hence strategies aimedat treating MS should not neglect the targeting and possible eliminationof MOG-specific B cell clones. While transfer of MOG-specific T-cellshas no apparent consequences in the recipient animal, co-transfer ofT-cells and antibodies against MOG can induce EAE in animal models,resembling multiple sclerosis in humans (Stefferl (1999) J. Immunol.163, 40-49). These findings suggest that auto-antibodies against MOGplay a central role in disease development and progression. Gold-labeledMOG peptides have been shown to bind specifically to disintegratingmyelin around axons in lesions of acute multiple sclerosis and inMOG-induced EAE in the marmoset model for MS, providing direct evidencethat autoantibodies against MOG mediate target membrane damage incentral nervous system demyelinating disease (Genain (1999) NatureMedicine, 5, 170-175; Raine (1999), Ann. Neurol. 46, 144-160). In acomparative study, MOG antibodies were shown to be common in patientswith multiple sclerosis (Reindl (1999) Brain, 122, 2047-2056).Therefore, and due to the central role of MOG-specific B-cells in MS ina further embodiment of the present invention, the composition of thepresent invention is particularly useful for the selective eliminationof these autoreactive lymphocytes (see herein below).

Cytochromes P450 (CYPs) and UDP-glucuronosyltransferases (UGTs) aretargets of autoantibodies in several hepatic and extrahepatic autoimmunediseases (Obermayer-Straup (2000) Can. J Gastroenterol 14, 429-39).

A number of Golgi proteins have been described as autoantigens,recognized by sera from patients with various forms of rheumaticdiseases. Examples of Golgi autoantigens are golgin-67, golgin-95/gml30, golgin-1 60, giantin, golgin-97, p230, golgin245 and p210(Eystathioy, (2000) J Autoimmun 14, 179-187; Mancini,. (2000) J CellBiol 149, 603-12, Linstedt (1993) Mol Biol Cell 4, 679-93; Griffith(1997) Arthritis Rheum 40, 1693-1702; Erlich (1996) J Biol Chem 271,18328-18337; Fritzler (1995) J Biol Chem 270, 31262-31268; Fritzler(1993) J Exp Med. 178, 49-62; Fritzler (1984) J Immunol. 132, 2904-2908;Rios (1994) J Cell Biol 125, 997-1013; Renier (1994) J Autoimmun 7,133-143).

In yet a further embodiment, the present invention provides for aninventive (poly)peptide, wherein said proteins sharing sequencesimilarity with microbial antigens or dietary proteins/components areselected from the group consisting of antigens mimicking proteins,polypeptides and/or carbohydrate structures from Streptococcus,Klebsiella, Proteus, M. tuberculosis, adenovirus, poliovirus, measlesvirus, retrovirus, papilloma virus, gluten and/or butyrophilin.

Microbial antigens can share regions of amino acid sequences homologywith mammalian proteins. Said microbial antigens can, therefore, elicitan autoimmune response, being an example of antigenic mimicry. Thecomposition of the present invention is, inter alia, useful fortreating, preventing and/or ameliorating autoimmune responses due tosuch an antigenic mimicry of microbial organisms. Examples of exogenousantigenic mimicry are known in the art and, inter alia, described inPaul, “Fundamental Immunology”, Raven Press, 1989.

For example, acute rheumatic fever is caused by antibodies againststreptococcal cell-wall antigens that cross-react with cardiac muscleand lead to arthritis, myocarditis and late scarring of heart valves(Khanna (1997) J. Autoimmun. 10, 99-106; Bronze (1993) J. Immunol. 151,2820-2828; Quinn. (1998) Infect Immun 66, 4418-4424). As discussedherein above, antibodies directed against the Sa antigen (streptococcalantigen) have been described in rheumatoid arthritis (Depres (1994),loc. cit.).

Furthermore, cross-reactivities of autoimmune antibodies exist betweenHLA-B27 and some Klebsiella strains, in particular of Klebsiellapneumoniae (see, inter allia, Dominguez-Lopez (2000) J. Rheumatol. 27,1453-1460). These cross-reactivities lead to ankylosing spondylitis.

Another example of antigenic mimicry which leads to autoimmunereactions, especially in rheumatoid arthritis, is the cross-reaction toHLA-DR antigens with proteins/(poly)peptides of Proteus mirabilis (see,eg. Ebringer (1992), Ann. Rheum. Dis. 51, 1245-6 or Ebringer (2000), JMed Microbiol 49, 305-11).

Furthermore, an autoimmune pathogenesis of atherosclerosis is describedand a cross-reactivity with human heat shock protein 60 (hsp60),expressed by endothelial cells, is involved in said autoimmune disease(Wick (2000), Herz 25,87-90).

In addition, the measles virus P3 protein resembles the above describedautoantigen MBP and may elicit EAE.

In a further embodiment, the present invention provides for a(poly)peptide as described herein above, when said dietary component isgluten or butyrophilin. In context of the present invention, dietarycomponents are nutrients that share structural or sequence similaritywith mammalian and/or human proteins or post-translational modificationsof human proteins, like N— or O-linked glycans. Such dietary componentscan cause molecular mimicry and induce an autoimmune reaction. Examplesof dietary components are gluten and the milk constituent butyrophilin.Celiac disease, also referred to as gluten sensitive enteropathy ischaracterized by IgA autoantibodies against anti-tissue transglutaminaseand antiendomysial antibodies (EMA). (Lock, J Clin Pathol 52 (1999),274-277; Rose, J Am Acad Derm 41 (1999) 957-961; Vitoria, J PediatrGastro Nutr 29 (1999), 571-574; Schuppan, Gastroent 119 (2000),234-242). Butyrophilin shares sequence homology to MOG. Butyrophilin hasbeen shown to modulate animal models of multiple sclerosis due tomolecular mimicry with MOG (Stefferl, J Immunol 165 (2000), 2859-2865).

The present invention also relates to a (poly)peptide wherein saidprotein of intercellular structures as described herein above isselected from the group consisting of desmoglein-1 (Dsg1), desmoglein-3(Dsg3), desmocollin, desmoplakin, envoplakin, periplakin, BPAG-1 (BP230;Liu, J. Dermatol. 2001, 28,647-650), BPAG-2 (BP180; Liu, loc. cit.) andHD1/plectin.

Pemphigus vulgaris and pemphigus foliaceus are caused by antibodiesagainst keratinocyte adhesion molecules desmoglein 3 (Dsg3) anddesmoglein 1 (Dsg1), respectively (Amagai, (1991) Cell 67, 869-877;Allen (1993), J Invest Dermatol 100, 685-91).

Other desmosomal or hemi-desmosomal proteins have been implied inpemphigus-related autoimmune diseases, in addition to desmogleins, alsoantibodies against desmoplakin, desmocollin, envoplakin, periplakin havebeen reported (Gooptu, (1999) Br. J. Dermatol. 141, 882-886; Chorzelski(1999) J Am Acad Dermatol 41, 393-400; Anhalt (1999) J. Am. Acad.Dermatol. 5, 763-6). Additional autoantigens are bullous pemphigoidantigens 1 (BPAG 1) and 2 (BPAG 2), BP230 (Schmidt, (2000) Arch Dermatol136, 174-178; Michelson (2000) J Histochem Cytochem 48, 535-544;Schuhmann (2000) Am J Pathol 156, 685-95; Dopp (2000) J. Am. Acad.Dermatol. 42, 577-583).

HD1/plectin, another member of the plakin family, has been described tobe recognized by sera from PNP (paraneoplastic pemphigus) patients(Proby (1999), J. Invest. Derm. 112,153-156)

In a yet more preferred embodiment, the present invention relates to a(poly)peptide, as described herein above wherein said T cells arecytotoxic T cells. It is of note that the present invention provides forinventive (poly)peptide constructs wherein a first domain comprising thedescribed de-immunized autoantigen or (a) fragment(s) thereof is adomain which suppresses and/or eliminates T-cell activation. Incontrast, the second domain of the inventive (poly)peptide constructcomprises an effector molecule which may, inter alia, specificallytrigger T-cell responses, for example the response of cytotoxic T-cells.In another embodiment, the invention provides for a compositioncomprising the (poly)peptides as defined herein above or thepolynucleotides, vectors or hosts as described herein below. Thecomposition is particularly useful for selective elimination ofautoreactive B-cells.

Furthermore, the present invention relates to a (poly)peptide orcomposition comprising at least one inventive (poly)peptide constructconsisting of at least two domains wherein one of said domains of saidconstruct comprises an autoreactive antigen or (a) fragment(s) thereofspecifically recognized by the Ig receptors of said autoreactive B-cellsand wherein one of said domains comprises an effector molecule capableof interacting with and/or of activating NK-cells, T-cells, macrophages,monocytes and/or granulocytes, wherein said effector molecule is areceptor-ligand or the Fc-part of an immunoglobulin. It is particularlypreferred that said effector molecule specifically binds to a moleculeof the CD3-receptor complex. It is even more preferred that saidreceptor-ligand is an antibody or (a) fragment(s) or derivative thereofor an aptamer.

In accordance with the present invention the term “antibody” relates tomonoclonal or polyclonal antibodies. Polyclonal antibodies (antiserum)can be obtained according to conventional protocols. Antibody fragmentsor derivatives comprise F(ab′)₂, Fab, Fv or scFv fragments; see, forexample, Harlow and Lane, “Antibodies, A Laboratory Manual”, CSH Press1988, Cold Spring Harbor, N.Y. Furthermore, in accordance with thepresent invention, the derivatives of the antibodies can be produced bypeptidomimetics. In the context of the present invention, the term“aptamer” comprises nucleic acid aptamers such as RNA, ssDNA (ss=singlestranded), modified RNA, modified ssDNA or PNAs which bind a pluralityof target sequences having a high specificity and affinity. Nucleic acidaptamers are well known in the art and, inter alia, described inFamulok, Curr. Op. Chem. Biol. 2 (1998), 320-327. The preparation ofaptamers is well known in the art and may involve, inter alia, the useof combinatorial RNA libraries to identify binding sites (Gold, Ann.Rev. Biochem. 64 (1995), 763-797). Said other receptors may, forexample, be derived from said antibody etc. by peptidomimetics.

In a particular preferred embodiment of the present invention, the abovementioned antibody derivative is a scFv directed against a molecule ofthe CD3 receptor complex.

In accordance with the present invention, the term “molecule of the CD3receptor complex” comprises any invariable proteins and or fragmentsthereof, which comprise CD3α, CD3β, CD3γ, CD3δ, CD3ε and CD3ζ (CD3α andCD3β are also known as TCRα and TCRβ). It is particularly preferred thatsaid scFv is directed against the CD3ε chain of the T-cell receptorcomplex. Single chain constructs comprising such a specificity are knownin the art and, inter alia, described in Mack (1997), J. Immunol. 158,3965-3970 or in the appended illustrative examples.

In a most preferred embodiment, the present invention relates to a(poly)peptide construct consisting of at least two domains wherein oneof said domains comprises a de-immunized, autoreactive antigen or (a)fragment(s) thereof specifically recognized by the Ig receptors of saidautoreactive B-cells and wherein a/the other domain comprises aneffector molecule capable of interacting with and/or of activatingNK-cells, T-cells, macrophages, monocytes and/or granulocytes, whereinsaid domain comprising a de-immunized, autoreactive antigen or (a)fragment thereof is de-immunized MOG or (a) fragment(s) thereof andwherein said domain comprising an immunological effector molecule is ananti-CD3 receptor or an Fc-part of an immunoglobulin.

The (poly)peptides and compositions as disclosed herein are inparticular useful for the elimination of autoreactive B-cells in vitro,in vivo and ex vivo. The appended examples illustrate that not only invitro depletions but also ex vivo as well as in vivo depletions areenvisaged with the composition of the present invention. In addition, itis illustrated that the present invention also provides for compositionswhich may be employed for the reduction of auto-reactive,antigen-specific immunoglobulins. Furthermore, the examples illustratethe generation of illustrative constructs comprising de-immunizedantigens or (a) fragment(s) thereof. Such examples comprise de-immunizedMOG and de-immunized AchR, in particular AchR alpha-chain. It isenvisaged that, should an antigen (auto-antigen) comprise more than oneT-cell epitope to be removed and/or substituted, preferably at least oneof these T-cell epitopes, preferably two, most preferably all T-cellepitopes are removed, substituted and/or eliminated. Yet, the inventiveconstruct still comprises an autoantigen or an autoantigen fragmentwhich is capable of specifically interacting with autoreactive B-cells.The interaction of the inventive constructs with B-cells may be testedby methods known in the art and such methods are illustrated in theappended examples.

The above mentioned (poly)peptides or compositions are particularlyuseful in treating, preventing and/or ameliorating autoimmune diseases,like multiple sclerosis. As described herein above, MOG is one of themajor autoantigens involved in multiple sclerosis.

The protein and/or nucleotide sequence of MOG are known to the personskilled in the art and described, inter alia, in WO 95/06727 or U.S.Pat. No. 5,532,351. Approaches to affect the T cell arm of multiplesclerosis include induction of tolerance using synthetic peptidesagainst the activation of MOG-specific T-helper cells ( see, WO95/07096, WO 96/12737, WO 97/35879, WO 99/12966). However, theseapproaches do not influence, ameliorate and/or modify the B-cell relatedsymptoms of the autoimmune reaction in multiple sclerosis.

The above described MOG belongs to the family of B7 homologous proteins,sharing the membrane topology and the extracellular immunoglobulindomain (Johns, (1999) J. Neurochem. 72, 1-9). Several human homologuesto MOG have been identified, but none of them has been reported to be anauto-antigen in MS. One homologue to MOG has been described that isexpressed in B lymphocytes (WO 98/33912). Furthermore, fusion proteinsof MOG or MOG homologues with Fc portions have been described (WO98/33912, WO 99/23867), yet, said Fc-portion fusion proteins have beendesigned to enhance protein expression and for use as an affinity tagfor purification. Often a protease site is introduced to remove the Fcportion after purification. This approach has been described in EP 0 464533. The above recited approaches of MOG-Fc fusion proteins have neitherbeen proposed nor envisaged the uses and compositions described herein,namely for the elimination of autoreactive B-cells via binding, interalia, to the B cell receptor and interaction with, e.g. complement or anFc-receptor bearing effector cell.

The present invention relates in a further embodiment to a (poly)peptide construct encoded by (a) a polynucleotide comprising a nucleicacid molecule encoding the polypeptide as depicted in SEQ ID NO. 28; (b)a polynucleotide comprising a nucleic acid molecule having the DNAsequence as depicted in SEQ ID NO. 27; (c) a polynucleotide hybridizingto a sequence which is complementary to a nucleotide sequence of (a) or(b); or (d) a nucleotide sequence being degenerate to the sequence ofthe nucleotide sequence of (c).

The above mentioned polynucleotide may be a naturally occurring nucleicacid molecule as well as a recombinant nucleic acid molecule. Saidpolynucleotide/nucleic acid molecule may, therefore, be of naturalorigin, synthetic or semi-synthetic.

It is also immediately evident to the person skilled in the art thatregulatory sequences may be added to the nucleic acid molecule of theinvention. For example, promoters, transcriptional enhancers and/orsequences which allow for induced expression of the above describedpolynucleotide may be employed. A suitable inducible system is forexample tetracycline-regulated gene expression as described, e.g., byGossen and Bujard (Proc. Natl. Acad. Sci. USA 89 (1992), 5547-5551) andGossen et al. (Trends Biotech. 12 (1994), 58-62).

The above described polynucleotide/nucleic acid molecules may either beDNA or RNA or a hybrid thereof.

With respect to the polynucleotides/nucleotide sequences characterizedunder (c) above, the term “hybridizing” in this context is understood asreferring to conventional hybridization conditions, preferably such ashybridization in 50% formamide/6×SSC/0.1% SDS and 100 μg/ml ssDNA, inwhich temperatures for hybridization are above 37+ C. and temperaturesfor washing in 0.1×SSC/0.1% SDS are above 55° C. Most preferably, theterm “hybridizing” refers to stringent hybridization conditions, forexample such as described in Sambrook., “Molecular Cloning: A LaboratoryManual”, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NewYork, 1989.

“Stringent hybridization conditions” refer, i.e. to an overnightincubation at 42° C. in a solution comprising 50% formamide, 5×SSC (750mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and 20 μg/ml denatured,sheared salmon sperm DNA, followed by washing the filters in 0.1×SSC atabout 65° C. Also contemplated are nucleic acid molecules that hybridizeto the polynucleotides of the invention at lower stringencyhybridization conditions. Changes in the stringency of hybridization andsignal detection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency); salt conditions, or temperature. For example, lowerstringency conditions include an overnight incubation at 37° C. in asolution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH₂PO4; 0.02M EDTA,pH 7.4), 0.5% SDS, 30% formamide, 100 μg/ml salmon sperm blocking DNA;followed by washes at 50° C. with 1×SSPE, 0.1% SDS. In addition, toachieve even lower stringency, washes performed following stringenthybridization can be done at higher salt concentrations (e.g. 5×SSC). Itis of note that variations in the above conditions may be accomplishedthrough the inclusion and/or substitution of alternate blocking reagentsused to suppress background in hybridization experiments. Typicalblocking reagents include Denhardt's reagent, BLOTTO, heparin, denaturedsalmon sperm DNA, and commercially available proprietary formulations.The inclusion of specific blocking reagents may require modification ofthe hybridization conditions described above, due to problems withcompatibility.

Furthermore, said polynucleotide/nucleic acid molecule may contain, forexample, thioester bonds and/or nucleotide analogues. Said modificationsmay be useful for the stabilization of the nucleic acid molecule againstendo- and/or exonucleases in the cell. Said nucleic acid molecules maybe transcribed by an appropriate vector containing a chimeric gene whichallows for the transcription of said nucleic acid molecule in the cell.

The polynucleotide/nucleic acid molecule of the present invention may bea recombinantly produced chimeric nucleic acid molecule comprising anyof the aforementioned nucleic acid molecules either alone or incombination. Preferably, the nucleic acid molecule of the invention ispart of a vector.

In a particularly preferred embodiment, (poly)peptide construct of thepresent invention is a (poly)peptide comprising the amino acid sequenceas depicted in SEQ ID NO. 28. Furthermore, (poly)peptide constructs areenvisaged, wherein the sequence as shown in SEQ ID No. 28 comprises atleast one modification. It is envisaged that that modification isselected so that the de-immunized auto-antigen still retains its bindingcapacity to autoreactive B-cells.

Said modification(s) may be selected from the group consisting of aminoacid exchange(s), insertion(s), deletion(s), addition(s),substitution(s), inversion(s) and duplication(s). Said modification(s)also comprise conservative and/or homologue amino acid exchange(s). Forexample, guidance concerning how to make phenotypically/functionallysilent amino acid substitution is given in Bowie (1990), Science 247,1306-1310. Moreover, tolerated conservative amino acid substitutionsinvolve replacement of the aliphatic or hydrophobic amino acids Ala,Val, Leu and IIe; replacement of the hydroxyl residues Ser and Thr;replacement of the acidic residues Asp and Glu; replacement of the amideresidues Asn and Gin; replacement of the basic residues Lys, Arg, andHis; replacement of the aromatic residues Phe, Tyr, and Trp, andreplacement of the small-sized amino acid substitution, variants ofpeptides of this invention include (i) substitutions with one or more ofthe non-conserved amino acid residues, where the substituted amino acidresidues may or may not be one encoded by the genetic code, or (ii)substitution with one or more of amino acid residues having asubstituent group, or (iii) fusion of the mature (poly)peptide constructwith another compound, such as a compound to increase the stabilityand/or solubility of the polypeptide (for example, polyethylene glycol),or (iv) fusion of the (poly)peptide construct with additional aminoacids, or leader or secretory sequence, or a sequence facilitatingpurification. Such variant (poly)peptide construct are deemed to bewithin the scope of those skilled in the art from the teachings herein.

Thus, the present invention also relates to peptides which are at least60%, more preferably at least 70%, more preferably at least 80%, morepreferably 90%, more preferably at least 95% and most preferably 99%identical or homologous to the (poly)peptide construct as shown in SEQID NO: 2 or 4. Specific strategies for obtaining (poly)peptideconstructs described herein above are known in the art. These methodscomprise recombinant and biochemical methods and are, inter alia,disclosed in Sambrook, loc. cit. Said methods also comprise proteinengineering or direct synthesis.

Yet, in a further embodiment, the present invention relates to apolynucleotide encoding at least one (poly)peptide construct asdescribed herein. The embodiments described herein above may be applied,mutatis mutantis, for the polynucleotide/nucleic acid molecule encodingsaid at least one (poly)peptide construct. It is understood thatcompositions as described herein and comprising at least onepolynucleotide encoding at least one (poly)peptide construct asdescribed herein may also be employed for the selective reduction ofautoreactive, antigen-specific immunoglobulins.

Additionally, in a more preferred embodiment the polynucleotide/nucleicacid molecule of the invention is part of a vector. In a particularlypreferred embodiment said vector is an expression vector.

Said vector of the present invention may be, e.g., a plasmid, cosmid,virus, bacteriophage or another vector used e.g. conventionally ingenetic engineering, and may comprise further genes such as marker geneswhich allow for the selection of said vector in a suitable host cell andunder suitable conditions. Particularly preferred vectors are vectorsas, inter alia, described in the appended examples and comprise, e.g.the expression vector CD19×CD3 pEF-dhfr.

Furthermore, the vector of the composition of the present invention, mayin addition to the polynucleotides/nucleic acid sequences describedherein above, comprise expression control elements, allowing properexpression of the coding regions in suitable hosts. Such controlelements are known to the artisan and may include a promoter, a splicecassette, translation initiation codon, translation and insertion sitefor introducing an insert into the vector. Preferably, the nucleic acidmolecule of the invention is operatively linked to said expressioncontrol sequences allowing expression in eukaryotic or prokaryoticcells.

Control elements ensuring expression in eukaryotic and prokaryotic cellsare well known to those skilled in the art. As mentioned herein above,they usually comprise regulatory sequences ensuring initiation oftranscription and optionally poly-A signals ensuring termination oftranscription and stabilization of the transcript. Additional regulatoryelements may include transcriptional as well as translational enhancers,and/or naturally-associated or heterologous promoter regions. Possibleregulatory elements permitting expression in for example mammalian hostcells comprise the CMV-HSV thymikine kinase promoter, SV40, RSV-promoter(Rous sarcoma virus), human elongation factor 1α-promoter, enhancers,like CMV enhancer or SV40-enhancer. For the expression in prokaryoticcells, a multitude of promoters including, for example, thetac-lac-promoter or the trp promoter, has been described. Besideselements which are responsible for the initiation of transcription suchregulatory elements may also comprise transcription termination signals,such as SV40-poly-A site or the tk-poly-A site, downstream of thepolynucleotide. In this context, suitable expression vectors are knownin the art such as Okayama-Berg cDNA expression vector pcDV1(Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (Invitrogene), pSPORT1, pEF-dhfr orprokaryotic expression vectors, such as lambda gt11, pDS or pET. Besidethe nucleic acid described herein, the vector may further comprisenucleic acid sequences encoding for secretion signals. Such sequencesare well known to the person skilled in the art. Furthermore, dependingon the expression system used leader sequences capable of directing thepeptides of the invention to a cellular compartment may be added to thecoding sequence of the nucleic acid molecules of the invention and arewell known in the art. The leader sequence(s) is (are) assembled inappropriate phase with translation, initiation and terminationsequences, and preferably, a leader sequence capable of directingsecretion of translated protein, or a protein thereof, into theperiplasmic space or extracellular medium. Optionally, the heterologoussequence can encode a fusionprotein including an C— or N-terminalidentification peptide imparting desired characteristics, e.g.,stabilization or simplified purification of expressed recombinantproduct. Once the vector has been incorporated into the appropriatehost, the host is maintained under conditions suitable for high levelexpression of the nucleotide sequences, and, as desired, the collectionand purification of the peptide(s) or fragments thereof of the inventionmay follow.

As mentioned herein above, the vector of the present invention may alsobe an expression vector. Gene therapy, which is based on introducingtherapeutic genes into cells by ex-vivo or in-vivo techniques is one ofthe most important applications of gene transfer. Suitable vectors,methods or gene-delivering systems for in-vitro or in-vivo gene therapyare described in the literature and are known to the person skilled inthe art; see, e.g., Giordano, Nature Medicine 2 (1996), 534-539;Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256 (1992),808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ. Res. 77(1995), 1077-1086; Onodua, Blood 91 (1998), 30-36; Verzeletti, Hum. GeneTher. 9 (1998), 2243-2251; Verma, Nature 389 (1997), 239-242; Anderson,Nature 392 (Supp. 1998), 25-30; Wang, Gene Therapy 4 (1997), 393400;Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO 97/00957; U.S.Pat. No. 5,580,859; U.S. Pat. No. 5,589,466; U.S. Pat. No. 4,394,448 orSchaper, Current Opinion in Biotechnology 7 (1996), 635-640, andreferences cited therein. In particular, vectors and/or gene deliverysystems are also described in gene therapy approaches in immunology orin neurology, for example Linden, Proc. Natl. Acad. Sci. U.S.A. 93(1996), 11288-11294; Maass, Hum. Gene Ther. 9 (1998), 1049-1059; Hallek,Cytokines Mol. Ther. 2 (1996), 69-79; Peel, Neurosci. Methods 98 (2000),95-104; Chen, J. Neurosci. Res. 55 (1999), 504-513. The nucleic acidmolecules and vectors described herein and comprised in the compositionof the present invention may be designed for direct introduction or forintroduction via liposomes, viral vectors (e.g. adenoviral, retroviral),electroporation, ballistic (e.g. gene gun) or other delivery systemsinto the cell. Additionally, a baculoviral system can be used aseukaryotic expression system for the nucleic acid molecules of theinvention. As documented in the appended examples, (poly) peptideconstructs comprised in the composition of the present invention mayalso be expressed in mammalian expression systems, for example inCHO-cells.

In a further embodiment, the present invention provides for acomposition comprising at least one (poly)peptide, at least one(poly)nucleotide, at least one vector and/or at least one host of thepresent invention. Preferably, said composition is a pharmaceuticalcomposition.

The term “composition”, in context of this invention, comprises at leastone (poly) peptide construct as defined herein, at least one (poly)nucleotide comprising a nucleic acid molecule encoding for such one(poly) peptide construct at least one vector comprising said(poly)nucleotide or at least one host comprising said (poly)nucleotide.Said composition, optionally, further comprises other molecules, eitheralone or in combination, like e.g. molecules which are capable ofmodulating and/or interfering with the immune system. The compositionmay be in solid, liquid or gaseous form and may be, inter alia, in aform of (a) powder(s), (a) tablet(s), (a) solution(s) or (an)aerosol(s). In a preferred embodiment, said composition comprises atleast two, preferably three, more preferably four, most preferably(poly)peptide constructs (and/or nucleic acid molecules encoding saidconstructs) as described in the invention.

The term “at least one (poly)peptide construct” as employed herein aboverelates to at least one (poly)peptide construct, at least two, at leastthree, at least four or at least five (poly)peptide constructs which maybe comprised in the composition of the present invention. The sameapplies, mutatis mutantis, for the (poly)nucleotide(s), the vector(s) orthe host(s) of the invention comprised in said composition.

The composition of the present invention may be employed for theselective elimination of autoreactive B-cells, preferably the selectiveelimination of such cells in individuals suffering from an autoimmunedisorder. Preferably, said individual is a human patient.

As will be discussed herein below, the composition of the presentinvention comprising nucleic acid molecule as described herein aboveand/or the above described vectors/hosts may be particularly useful intreating, preventing and/or ameliorating an autoimmune desease/disorder.Therefore, said compositions may be employed in gene therapy approaches.

For gene therapy applications, said nucleic acids encoding the(poly)peptide constructs as described herein may be cloned into a genedelivering system, such as a virus. Preferably, IL-4 carrying plasmidsmay be employed.

Therefore, it is particularly preferred that the composition of thepresent invention comprises a host transformed with the vector describedherein above. It is even more preferred that said host is a mammaliancell, most preferred is a human cell.

In yet a further embodiment, the present invention relates to acomposition as described herein above which further comprises a compoundcapable of selectively eliminating plasma cells and/or a compoundcapable of selectively eliminating (an) auto-antibody(ies). Preferably,said compound capable of selectively eliminating plasma cells is anantibody or (a) fragment(s) or a derivative thereof specificallydetecting an plasma cell-specific epitope. Even more preferably, saidcompound capable of selectively eliminating (an) auto-antibody(ies) isan anti-idiotypic antibody or (a) fragment(s) or a derivative thereofspecifically reacting with said auto-antibody(ies).

Furthermore, the present invention provides for compositions asdescribed herein for the selective reduction of autoreactiveimmunoglobulins/for the selective elimination of auto-antibody(ies). Asdocumented in the appended examples the constructs as disclosed hereinand employed in the compositions of the present invention are not onlycapable of selectively eliminating autoreactive B-cells but also ofreducing titers of autoreactive immunoglobulins. Preferably, saidselective reduction of autoreactive immunoglobulins leads to atiter-reduction of at least 20%, at least 40%, at least 50%, at least60%, most preferably at least 70%. Titers of autoreactiveimmunoglobulins may be measured by methods known in the art and as shownin the appended examples, e.g. the measurement of circulatingautoreactive immunoglobulins. Such methods, e.g., methods of detectingautoantibodies in sera comprise ELISA-tests, RIA, immunodiffusions,immunoprecipitations, Western blotting, affinity-chromatographie.Furthermore, in situ-methods are envisaged which comprise the use oflabeled peptide, preferably immunogold-labeled peptides andhigh-resolution microscopy.

In a most preferred embodiment of the present invention, the compositionof the present invention is a pharmaceutical composition optionallycomprising a pharmaceutically acceptable carrier, as discussed hereinabove.

The pharmaceutical composition of the present invention may beparticularly useful in preventing, ameliorating and/or treatingautoimmune disorders/diseases, as described herein above and hereinbelow.

Examples of suitable pharmaceutical carriers, excipients and/or diluentsare well known in the art and include phosphate buffered salinesolutions, water, emulsions, such as oil/water emulsions, various typesof wetting agents, sterile solutions etc. Compositions comprising suchcarriers can be formulated by well known conventional methods. Thesepharmaceutical compositions can be administered to the subject at asuitable dose. Administration of the suitable compositions may beeffected by different ways, e.g., by intravenous, intraperitoneal,subcutaneous, intramuscular, topical, intradermal, intranasal orintrabronchial administration. It is particularly preferred that saidadministration is carried out by injection and/or delivery, e.g., to asite in a brain artery or directly into brain tissue. The compositionsof the invention may also be administered directly to the target site,e.g., by biolistic delivery to an external or internal target site, likethe brain. The dosage regimen will be determined by the attendingphysician and clinical factors. As is well known in the medical arts,dosages for any one patient depends upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Proteinaceouspharmaceutically active matter may be present in amounts between 1 ngand 10 mg per dose; however, doses below or above this exemplary rangeare envisioned, especially considering the aforementioned factors. Ifthe regimen is a continuous infusion, it should also be in the range of1 ug to 10 mg units per kilogram of body weight per minute,respectively. In context of the present invention, it is preferred thatthat the peptides of the present invention are employed inconcentrations of less than 500 μg/ml, more preferred at less than 100μg/ml, more preferred of less than 10 μg/ml and most preferred of lessthan 1 μg/ml.

Progress can be monitored by periodic assessment. The compositions ofthe invention may be administered locally or systemically. Preparationsfor parenteral administration include sterile aqueous or non-aqueoussolutions, suspensions, and emulsions. Examples of non-aqueous solventsare propylene glycol, polyethylene glycol, vegetable oils such as oliveoil, and injectable organic esters such as ethyl oleate. Aqueouscarriers include water, alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclesinclude sodium chloride solution, Ringer's dextrose, dextrose and sodiumchloride, lactated Ringer's, or fixed oils. Intravenous vehicles includefluid and nutrient replenishers, electrolyte replenishers (such as thosebased on Ringer's dextrose), and the like. Preservatives and otheradditives may also be present such as, for example, antimicrobials,anti-oxidants, chelating agents, and inert gases and the like.Furthermore, the pharmaceutical composition of the invention maycomprise further agents depending on the intended use of thepharmaceutical composition. Said agents may be drugs acting on theimmune system, like FK506, cyclosporin, IFNbeta, azathioprine,cyclophosphamide, prednisone, corticosteroids, cyclosporin A,calcineurin, rapamycin and neuroprotective agents such as theneurotrophins (NGF, BDNF, NT-3); see also Webster, Mult. Scler. 3(1997), 113-120; Ebadi, Neurochem. Int. 30 (1997), 347-374.

The compositions, also the pharmaceutical compositions, of the presentinvention may be tested for functionality by different approaches. Forexample, as a surrogate test system several approaches can be chosen: 1)Establishment of a B-cell derived cell line expressing a membrane-bound1 g derived from a murine hybridoma cell line specific for theauto-antigen, 2) Hybridoma cells expressing membrane-bound 1 g could beisolated using auto-antigen; 3) Alternatively, autoreactive B cells maybe derived from transgenic mice with autoreactive B cells. Mouse modelswhich may be employed are known in the art. For example, Litzenburger(see J. Exp. Med. (1998) 188, 169-180) has establised such a mouse modelfor multiple sclerosis using the DNA sequences of the heavy chainvariable domain encoding the 8.18-C5 antibody to MOG. Further in-vivomodels for B-cell dominant autoimmune diseases have been reviewed inMurakami and Tasuku (Curr. Opin. Immunol. (1997) 9, 846-850),Christadoss et al. (Clin. Immunol. (2000) 94, 75-87). Experimentalautoimmune encephalomyelitis (EAE) in rats and marmorsets can be used asa surrogate model for multiple sclerosis ('t Hart et al. (2000) Immunol.Today 21, 290-297; Stefferl et al. (1999) J. Immunol. 163, 4049). It isalso envisaged that the above mentioned functionality approach comprisesthe measurement of circulating immunoglobulins and in particular ofautoreactive immunoglobulins after administration of a composition asdefined herein. Such a measurement is shown in the appended examples andmay easily be employed in samples from test animals, as well as insamples of humans. Preferably, said sample is a blood sample orcerebrospinal fluid.

The present invention relates in another embodiment to the use of atleast one (poly)peptide construct as defined herein, of at least onepolynucleotide described herein above or encoding at least one(poly)peptide construct as defined herein or of at least one vectordescribed herein above for the preparation of a pharmaceuticalcomposition for the treatment, amelioration and/or prevention of anautoimmune disease, preferably of a human autoimmune disease. Saidpharmaceutical composition may be useful for the autoimmune diseases anddisorders mentioned herein above and are particularly useful for thetreatment, prevention or amelioration of diseases selected from thegroup consisting of Pemphigus vulgaris, Bullous pemphigoid,Goodpasture's syndrome, autoimmune haemolytic anemia (AIHA), rheumatoidarthritis, Systemic Lupus erythematosus, Grave's disease (autoimmunehyperthyroidism), contact dermatitis, Myasthenia gravis, juvenilediabetes, Sjögren's syndrome, autoimmune throiditis, primaryhypoadrenalism (Addison's disease), multiple sclerosis, thrombocytopenicpurpura, pemphigous foliaceous, linear IgA dermatosis, Morbus Wegener(granulomatosis) and celiac disease.

The present invention also provides for a method of therapy,amelioration and/or prevention of an autoimmune disease comprising theadministration to a subject in need of such therapy and/or prevention aneffective amount of at least one (poly)peptide construct as definedherein, of at least one polynucleotide as defined herein, of at leastone vector defined herein or of at least one host of described herein.It is particularly preferred that said method be employed for thetreatment and/or prevention of autoimmune disorders as described herein,like, but not limited to, Pemphigus vulgaris, Bullous pemphigoid,Goodpasture's syndrome, autoimmune haemolytic anemia (AIHA), rheumatoidarthritis, Systemic Lupus erythematosuas, Grave's disease (autoimmunehyperthyroidism), contact dermatitis, Myasthenia gravis, juvenilediabetes, Sjogren's syndrome, autoimmune throiditis, primaryhypoadrenalism (Addison's disease), multiple sclerosis thrombocytopenicpurpura, pemphigous foliaceous, linear IgA dermatosis, Morbus Wegener(granulomatosis) and celiac disease. It is particularly preferred thatthe subject to be treated by the method of the invention be a humansubject.

The compositions, in particular the pharmaceutical compositions, usesand methods of the invention can be used for all kinds of diseaseshitherto unknown as being related to or dependent on auto-antigensand/or the production of auto-antibodies. Said compositions, uses andmethods of the invention may be desirably employed in humans, althoughanimal treatment is also encompassed by the uses and methods describedherein.

In accordance with this invention, the terms “treatment”, “treating” andthe like are used herein to generally mean obtaining a desiredpharmacological and/or physiological effect. Said effect may beprophylactic in terms of completely or partially preventing a disease,in particular, an autoimmune disease, or a symptom thereof and/or may betherapeutic in terms of completely or partially curing a disease, inparticular, an autoimmune disease, and/or (an) adverse effect(s)attributed to said disease. The term “treatment” as used hereinincludes: (a) preventing the disease from occurring in a subject whichmay be predisposed to the disease but has not yet been diagnosed ashaving it; (b) inhibiting the disease, i.e. arresting its development;or (c) relieving the disease, i.e. causing regression of the disease.

Additionally, the (poly)peptides or compositions of the invention areparticularly useful in assays employing animal models for autoimmunediseases. Preferably, said animal models are mammalian (e.g. mousemodels). When employing the present invention with an animal model, itis preferred to use corresponding homologues of IgG (e.g. preferablymIgG2a for a mouse model).

The figures show:

FIG. 1 Overview of concept for the elimination of autoreactive B cells.

Overview of elimination of autoreactive B cells using moleculescomprising an autoantigen and an effector molecule domain. Abbreviationsare ECR: Effector cell receptor; ECRbp: Effector cell receptor bindingprotein; Ag: Antigen.

FIG. 2. Schematic representation of autoantigen—effector domainmolecules.

A. MOG×CD3 arrangement. B. MOG-Fc arrangement. MOG-ex depicts theextracellular domain of the MOG protein.

FIG. 3. Expression and purification of MOG×CD3.

MOG×CD3 was expressed in CHO cells and purified by cation exchange,followed by nickel chelate chromatography and gel filtration. (A) 5 Gelfiltration peaks are shown, separated by SDS-PAGE and analyzed byCoomassie-blue staining. Peak number 5 contains monomeric MOG×CD3protein of a molecular weight of approximately 47 kDa. (B) Gelfiltration peaks were separated on SDS-PAGE identical to A, transferredto a nitrocellulose membrane and incubated with an anti-MOG antibody(8.18-C5). Only peak 5 containing the 47 kDa monomeric MOG×CD3 reactswith monoclonal anti-MOG antibody (8.18-C5). Higher molecular weightbands present in peaks 1 to 4 are not recognized by anti-MOG antibody. Mindicates the molecular weight marker.

FIG. 4. Expression and purification of MOG-Fc fusion protein.

MOG-Fc fusion protein was expressed in CHO cells and purified byaffinity chromatography using Protein A sepharose. Purified protein wasseparated by SDS-PAGE and analyzed by coomassie-blue staining (A) in thepresence of the reducing agent DTT, or by Western blotting onnitrocellulose (B), followed by detection using a monoclonal antibodyagainst MOG (8.18-C5). As expected, MOG-Fc fusion protein has amolecular weight of approximately 50 kDa under reducing conditions(+DTT), but runs at a molecular weight of approximately 115 kDa in theabsence of DTT (−DTT), indicative for a dimer under non-reducingconditions. Detection in (B) was with an anti-Fc monoclonal antibody.

FIG. 5. Detection of MOG fusion protein in ELISA.

ELISA plates were coated with anti-MOG antibody 8.18-C5 to capture thefusion proteins by their MOG domain. (A) Bound MOG-Fc fusion protein wasdetected with an AP-conjugated anti-human Fc antibody. (B) MOG×CD3fusion protein was detected by an AP-labeled chicken versus single chainFv anti-CD3 polyclonal antibody. Absorption unit is indicated by AU.

FIG. 6. Binding of MOG-Fc fusion protein to B-cells of a transgenicmouse.

Resting B-lymphocytes (CD43⁻) were isolated and incubated with MOG-Fc.Bound fusion protein was detected by a FITC-labeled anti-humanFc-specific antibody. Only a subpopulation of transgenic B-cells expressMOG-reactive B-cell receptors.

FIG. 7. Binding of MOG×CD3 to human T-cells.

MOG×CD3 was incubated with human CD3-positive PBMCs, and bound fusionprotein was detected with monoclonal anti-MOG antibody followed byFITC-labeled anti-mouse IgGI antibody.

FIG. 8. 8.18-C5 hybridoma cells show surface expression of IgGI.Hybridomas were cultivated in serum-free medium for 4-5 months, andcell-surface IgG1 expression was examined by FACS analysis (A) withFITC-labelled anti-IgG1 antibody and (B) with biotinylated rMOG(recombinant MOG extracellular domain), followed by FITC-labeledstreptavidin.

FIG. 9. Cytotoxic activity of autoantigen-effector proteins.

FIG. 9 shows a FACS-based cytotoxicity assay with MOG×CD3 fusionprotein. 8.18-C5 target cells were incubated with human CD3+ PBMCs andserial dilutions of MOG×CD3 protein in RPMI/10% FCS. Incubation wasperformed for 16 h at 37° C./5% CO₂ After incubation, target cells werelabeled with anti-IgG1 antibody and analyzed for viability throughpropidium iodide staining. Viable murine IgG1 (+) cells were counted andexpressed as percentage of viable target cells in control: (A) MOG×CD3,(B) MOG-FC fusion protein.

FIG. 10. Specificity of elimination of autoreactive B cells.

MOG×CD3 cytotoxicity was measured with a FACS-based assay. Specifictoxicity was assayed as described in FIG. 9. To measure spontaneouslysis target and effector cells had been incubated separately for theduration of the assay and were combined just prior to analysis. MOG×CD3and MOG-Fc were used in a concentration of 10 μg/ml and incubated for 16hours. (A) MOG×CD3, (B) MOG-Fc.

FIG. 11. Cytotoxicity of MOG×CD3 is inhibited by recombinant MOG.MOG×CD3 was used at 0.1 μg/ml in a FACS-based cytotoxicity assay asdescribed for FIG. 9. Recombinant, biotinylated MOG was added to obtaina final concentration of 20 μg/ml.

FIG. 12. MOG×CD3 binds to 8.18-C5 target cells.

Binding was assessed with FITC-labeled anti-HIS antibody (Dianova) at 10μg/ml.

FIG. 13. Cytotoxic activity of MOG×CD3 is dependent on effector cell totarget ratio

Assay was performed as described for FIG. 9 with varying concentrationsof effector cells and a constant number of 8.18-C5 target cells.

FIG. 14. Flow cytometric analysis of cell binding properties of MOG-Fc.

(A) Specific binding of MOG-Fc fusion protein to IgM+ B-cells derivedfrom splenocytes from anti-MOG transgenic mice. Splenocytes from wt(upper panel) and anti-MOG transgenic mice (lower panel) were preparedand incubated with MOG-Fc. MOG-Fc bound to IgM-positive cells wasdetected by FITC-labeled human Fcy-specific antibody.

(B, C) Selective binding of MOG-Fc fusion protein to Mac1- andCD5-positive wild-type mouse splenocytes. Single-cell suspensions of wtsplenocytes were prepared. Cells were incubated with 50 μg/ml MOG-Fcprotein (lower panel) or PBS (upper panel). Cell-bound MOG-Fc wasdetected with biotinylated, monoclonal anti-MOG antibody 8.18-C5,followed by counterstaining with streptavidin-FITC, andMac1-phycoerythrin (PE) or CD5-PE.

(D) Specific binding of MOG-Fc fusion protein to a murine macrophagecell line. The monocyte/macrophage cell line p388.D1 expressing murineFc receptors was incubated with MOG-Fc. Bound MOG-Fc was detected viaFITC-labeled anti-human Fcγ antibody by flow cytometry (left panel).Binding was quantitated and expressed as mean fluorescence intensity(MFI) (right panel).

FIG. 15. Specificity of MOG-Fc mediated cytotoxicity

A) Effect of non-specific human IgG1.

8.18-C5 target cells were incubated with human PBMCs and 10 μg/ml MOG-Fcprotein in RPMI/10% FCS. Incubation was performed for 16 h at 37° C. and5% CO₂ . After incubation, target cells were labeled with anti-murineIgG1 antibody (mlgG) and analyzed for viability through propidium iodide(PI) staining. Column 1: incubation of PBMCs and target cells in theabsence of isotype control and MOG-Fc. Column 2: the effect of arecombinant human IgG1 (isotype control). Column 3: the effect of MOG-Fcunder identical assay conditions.

B) Effect of unrelated murine cell line expressing cell surface IgG1 ofnon-MOG specificity.

Mouse B cell line TIB-208 is expressing cell surface IgG1 of non-MOGspecificity.

Column 1: unspecific lysis of TIB-208 cells in the presence of humanPBMC.

Column 2: the effect of MOG-Fc (10 μg/ml) on the viability of TIB-208cells.

Column 3: the effect of MOG-Fc (10 μg/ml) on hybridoma line 8.18-C5under identical assay conditions. Error bars indicate S.D. values oftriplicates.

FIG. 16. Ex vivo depletion of B-cells from anti-MOG transgenic mice byMOG-Fc

Single cell suspensions of spleens from anti-MOG transgenic mice wereprepared. Splenocytes were cultured in the absence (dark columns) orpresence (white columns) of MOG-Fc (10 μg/ml). After incubation, the Bcell population was analyzed by flow cytometry using antibodies againstthe B cell markers IgM, IgD and CD19. The frequency of cell populationsis expressed as percentage of total live cells within the lymphocytegate. Error bars represent S.D. values of triplicates.

FIG. 17. In vivo depletion of MOG-reactive B cells by MOG-Fc intransgenic mice

Anti-MOG transgenic mice were left untreated (control) or treated i.p.with 100 μg of MOG-Fc protein on days 1 and 3.

(A) Flow cytometric analysis of B cells from MOG-Fc-treated anti-MOGtransgenic mice.

Upper panel, the effect of MOG-Fc on IgM^(a) (allotype a)-positive,rMOG-reactive B cells. Lower panel, analysis of MOG-Fc binding toB220-positive B cells as detected by an anti-human Fcy antibody.Peripheral blood lymphocytes (PBLs) were prepared 1 day post-treatmentand the MOG^(high) B-cell population was analyzed as indicated. FiveMOG-Fc-treated and three control animals were analyzed. Representativeresults are shown.

(B) The effect of MOG-Fc on the MOG^(high) B-cell population in anti-MOGtransgenic mice.

FACS data from five animals were quantitated and were shown aspercentage of total B220⁺ cells. Bars give S.D. values.

FIG. 18 In vivo depletion of anti-MOG reactive B cells in wildtype miceafter cellular transfer

1.5×10⁷ B cells derived from anti-MOG transgenic mice with BL/6background were transferred intraveniously into wt BL/6. Mice weredivided into 3 groups (N=5 per group) and treated with treated withMOG-Fc (100 μg each treatment) intraperitoneally 1, 2 and 3 dayspost-transfer (treated), with human IgG1 isotype (isotype control) andPBS (PBS).

24 h (A) and 72 h (B) after the last treatment, peripheral blood wascollected by tail bleeding and analyzed for anti-MOG reactive B cellsvia FACS staining using recombinant MOG-Fc and PE-conjugated anti-humanFc antibody (ICN).

One day post-treatment with MOG-Fc depletion of MOG-reactive B cells(FIG. 18A) was observed. This effect was more prominent three dayspost-treatment showing a depletion af autoreactive B-cells in the orderof 70% (FIG. 18B).

FIG. 19 Reduction of MOG-specific IgG in wildtype mice after cellulartransfer

1.5×10⁷ B cells derived from anti-MOG transgenic mice with BL/6background were transferred intraveniously into wildtype BL/6. Mice weredivided into 3 groups (N=5 per group) and treated with MOG-Fc, humanIgG1 isotype control and PBS. Peripheral blood was collected by tailbleeding 24 h (FIG. 19A) and 72 h (FIG. 19B) post-treatment and serumwas analyzed for anti-MOG specific IgG titers by ELISA.

FIG. 20: Proliferative response of M36 T-cell line to mutated M36peptides

Mutated versions of M36 peptide were synthesized. A panel of 15 peptides(D-Q) was generated, each carrying a single amino acid point mutation toalanine (Ala-Scanning). Murine and human M36 wildtype peptides (M36/mand M36/n, respectively) were used as controls. Proliferation ofM36-responsive T-cell line to peptide stimulation was assessed by 3-Hthymidine incorporation assay.

FIG. 21: Dose response of M36 T-cell line to M36 peptide

M36-specific mouse T cell line was stimulated with APCs derived from SJLmice loaded with human M36 peptide in decreasing concentrations ( 1/10,1/100, 1/1000, 0). Proliferation was assessed in a dose responseexperiment by standard 3-H thymidine incorporation assay.

FIG. 22: Peptide competition assay of mutated M36 peptides

Peptides identified as non-stimulatory for M36 T cells were furthertested for their ability to compete with wildtype M36 peptide.Irradiated splenocytes from SJL mice were used as APCs and loaded withwildtype M36 peptide (primary stimulation). Cells were washed withmedium, and Ala-substituted peptide variants (hu/5-hu/13) of wildtypehuman M36 peptide (hu/hu) were added for secondary stimulation as wellas M36 T cells. As control, medium only was used for stimulation (hu/0).Peptides substituted at positions 10 (hu/10), 12 (hu/12) or 13 (hu/1 3)of the M36 region were able to inhibit proliferation of M36 T cells.

FIG. 23: Binding of eMOG-Fc protein to B-cells from splenocytes of THmice versus negative wildtype (wt) controls

Flow cytometric analysis shows binding of eMOG-Fc to B-cells of Ig-MOGtransgenic TH mice. Splenocytes from wt (upper panel) and TH mice (lowerpanel) were prepared and incubated with eMOG-Fc. eMOG-Fc bound toIgM-positive cells was detected by FITC-labeled human Fcγ-specificantibody (huFc). Stainings show selective binding to transgenic B cells(panel A, C) in the absence of unselective binding to T cells. This wasdetected through the marker CD5 (panel B). No staining was detected withthe secondary anti-Fc antibody alone in the absence of eMOG-Fc fusionprotein (D).

FIG. 24: Ex vivo depletion of B-cells derived from TH mice by eMOG-Fc

Single cell suspensions of spleens from Ig-MOG transgenic TH mice wereprepared. Splenocytes were cultured with control human IgG1 protein ascontrol or eMOG-Fc (10 μg/ml). After incubation, the B-cell populationwas analyzed by flow cytometry using antibodies against the pan B-cellmarker CD19. The frequency of cell populations is expressed aspercentage of total live cells within the lymphocyte gate. Error barsrepresent S.D. values of triplicates.

The present invention is additionally described by way of the followingillustrative non-limiting examples, that provide a better understandingof the present invention and of its many advantages.

EXAMPLE 1 Expression Vectors for Autoantigen Fragment—αCD3 and −FcFusion Proteins

Expression of the construct of interest is driven by the promotor of thehuman elongation factor alpha (Kufer; PNAS 92 (1995): 7021). Thispromotor is known to be very efficient in virtually all eukaryoticcells, thereby making this expression system a powerful tool for highprotein expression without limitations regarding the selected eukaryotichost cell line. A versatile multiple cloning site (MCS) facilitates thecloning of the construct. The expression of the construct of interest islinked to the expression of the selection marker dihydrofolate reductase(DHFR) via the internal ribosomal entry site (IRES). This arrangementassures that almost all stably transfected cells will express theconstruct, as both genes depend on the promotor of EFα. A strongpolyadenylation signal for both genes is provided by the SV40polyadenylation site, and the pUC18 backbone of the plasmid provides awell-characterized plasmid backbone with ampicillin resistance forbacterial selection.

EXAMPLE 2 Construction of an exemplified antigenxeffector molecule; Anauto-antigen×αCD3 fusion protein: MOG×CD3

The expression vector pEF-dhfr CD19xCD3 (Loffler; Blood 95 (2000): 2098)was used as the basis for the construction of MOG×CD3 by removing theregion coding for the single-chain αCD19 antibody. As described below,the remaining αCD3 expression cassette was used for insertion of cDNAcoding for the auto-antigenic domain of the human MOG protein andprovides an ideal system for further cloning of auto-antigen×αCD3 fusionproteins (FIG. 2A).

2.1. Isolation of RNA from MOG-Transfected Fibroblasts and cDNASynthesis

Total RNA was isolated from MOG-transfected fibroblasts (Schluesener, J.Immunol. 139 (1987), 4016-4021) using the Qiagen RNEasy RNA ExtractionKit according to the manufacturer's suggestions. RNA was dissolved inH₂O and stored at −80° C. cDNA was synthesized as follows: 2 μg totalRNA were added to 0.5 μg Oligo-dT primer in a total reaction volume of12 μl. The reaction mixture was incubated at 70° C. for 10 min. Then, 4μl 5× First Strand Buffer (Gibco BRL), 2 μl 0.1M DTT and 1 μl 10 mMdNTPs were added. Incubation was performed at 42° C. for 2 min, afterwhich 200 U of Superscript II Reverse Transcriptase (RT) (Gibco BRL)were added. The reaction mixture was incubated for 50 min. at 42° C.Then, RT was inactivated due to a 15 min incubation step at 70° C.Isolated cDNA was stored at −20° C.

2.2. Amplification of MOG-Coding cDNA Fragments

The following primers were chosen to obtain the cDNA coding for theextracellular domain of the human MOG protein (MOG-ex): Primer 1 (MOG-Ex5′): 5′-TAGAATTCATGGCAAGCTTATCGAGACCC-3′ (Seq ID No 5) Primer 2 (MOG-Ex3′): 5′-CATCCGGATCCAGGGCTCACCCAGTAGA-3′ (Seq ID No 6)

Primers were designed to amplify the first 462 bases of the codingregion for the leader sequence and extracellular domain of the human MOGprotein. The primers contained EcoRI and BspEl restriction sites at 5′and 3′ ends, respectively. Polymerase chain reactions (PCR) conditionswere: 50 pmol primer, 1 μl dNTPs 10 mM, 4 μl cDNA, 5 μl Pfu-buffer 5×(Stratagene) and 5 U Pfu-Polymerase (Stratagene) in a final volume of 50μl. The final product of 474 bp was verified on a ethidium bromidestained 2% agarose gel.

2.3 Construction of MOG×CD3 fusion protein

The expression vector CD19×CD3 pEF-dhfr was digested with EcoRI andBspEI, leading to removal of the fragment coding for the anti-CD19 scFvdomain. The remaining vector-anti CD3 scFv portion was gel-extracted(gel extraction kit, Qiagen). Equally, MOG-ex was partially digestedwith BsaWI and EcoRI, and the corresponding fragment of 474 bp of lengthisolated via gel-extraction. BsaWI restriction was chosen due to thisenzyme's insensitivity to dam-methylation at the original BspEIrestriction site. DNA was eluted in 30 μl Tris pH 8.5 and stored at −20°C. Ligation of isolated DNA fragments was performed with equal volumesof extracted DNA and 5 U of T4 DNA Ligase in a total volume of 20 μl in1× T4 buffer (Roche Biochemicals) for 30 min. at room temperature (RT).Of each ligation reaction, 3 μl were used to transform E. coli XL-1 Blueas described above. Colonies were picked and subjected to MiniPrepanalysis. Following analytical restriction enzyme digestion, appropriateclones were sequenced (Sequiserve, Munich). Clones were grown in 300 mlLB-Amp medium, and plasmid DNA was isolated using the Qiagen plasmidprep kit. Corresponding nucleotide and amino acid sequences of thisconstruct are depicted and illustrated in SEQ ID NOs: 1 and 2.

EXAMPLE 3 Construction of Auto-Antigen—Fc fusion protein: MOG-Fc

3.1. Isolation of RNA from HD69-Transfected CHO Cells and cDNASynthesis.

Total RNA was isolated from HD69-transfected CHO cells (WO9846645) usingthe Qiagen RNEasy RNA Extraction Kit according to the manufacturer'ssuggestions. RNA was dissolved in H₂O and stored at −80° C.Complementary DNA synthesis was performed: briefly, 2 μg total RNA wereadded to 0.5 μg Oligo-dT primer in a total reaction volume of 12 μl. Thereaction mixture was incubated at 70° C. for 10 min. Then, 4 μl 5× FirstStrand Buffer (Gibco BRL), 2 μl 0.1M DTT and 1 μl 10 mM dNTPs wereadded. Incubation was performed at 42° C. for 2 min, after which 200 Uof Superscript II Reverse Transcriptase (RT) (Gibco BRL) were added. Thereaction mixture was incubated for 50 min. at 42° C. Then, RT wasinactivated due to a 15 min incubation step at 70° C. Isolated cDNA wasstored at −20° C.

3.2. Amplification of IgG1-Coding cDNA Fragments

In order to obtain cDNA coding for the Fc-domain of the human IgG1antibody backbone, the following primers were chosen: Primer 3 (IgG1-Fc5′): 5′-TATCCGGAGAGCCCACCTCTTGTGACAAAAC-3′ (Seq ID No 7) Primer 4(IgG1-Fc 3′): 5′-GTGTCGACTCATTTACCCGGAGACAGGG-3′ (Seq ID No 8)

Yet, an even more preferred primer (IgGl-Fc5′) is the following: Primer5 (IgG1-Fc5′): 5′-TATCCGGAGAGCCCAAATCTTGTGACAAAAC-3′ (SEQ ID NO:9)

Primers were designed to amplify the 699 bases coding for the Fc part ofthe human IgG1 backbone, while introducing BspEl and Sall restrictionsites at 5′ and 3′ ends of the amplified fragment, respectively.Amplification was performed according to standard PCR protocols.Briefly, 50 pmol/each of appropriate primer, 1 μl dNTPs 10 mM, 4 μlcDNA, 5 μl Pfu-buffer 5× (Stratagene) and 5 U Pfu-Polymerase(Stratagene) were added to a final volume of 50 μl in H2O. The finalproduct, containing the flanking restriction sites, was 711 bp inlength.

DNA was recovered from PCR reaction mixture according to themanufacturer's suggestions (Boehringer High Pure PCR ProductPurification Kit, cat. no. 1 732 676). Blunt-ended PCR productsgenerated by Pfu DNA polymerase were ligated into pCR-script vector(Stratagene #211188) according to manufacturers protocol (Stratagene).Plasmids were transformed into competent E. coli strain XL-1 Blue using4 μl of ligation product added to 50 μl of E. coli. The mixture wasincubated on ice for 10 min., 1 min. at 42° C., and then again on icefor 2 min. Thereafter, 150 μl LB-medium were added and expression ofampicillin resistance genes was induced due to 45 min at 37° C. whileshaking. Reaction mixtures were plated on LB-Amp Agarose plates (50 μgampicillin/ ml) and incubated at 37° C. for 16 h. Colonies were pickedand grown in LB-Amp medium (100 μg/ ml) for 8-12h. Bacteria were spundown, and plasmid DNA was isolated according to manufacturer'ssuggestions (Plasmid Mini-Kit, Qiagen). DNA was subjected to restrictionenzyme analysis, and suitable clones were sequenced (SequiServe,Munich). Correct clones were grown in 300 ml LB-Amp medium, and plasmidDNA was isolated according to manufacturer's instructions (PLasmid MaxiKit, Qiagen).

3.3. Construction of MOG-Fc Fusion Protein

In order to obtain the desired construct (FIG. 2B), expression vectorCD19×CD3 pEF-dhfr was subjected to restriction with EcoRI and Sall,leading to removal of the fragment coding for CD19×CD3. The remaininglinearized vector was gel-extracted (gel extraction kit, Qiagen).Equally, MOG-ex was partially digested with BsaWl and EcoRI, and thecorresponding fragment of 474 bp of length was isolated viagel-extraction as described above. BsaWl restriction was chosen due tothis enzyme's insensitivity to dam-methylation at the original BspElrestriction site. DNA was eluted in 30 μl Tris pH 8.5 and stored at −20°C. Ligation of isolated DNA fragments was performed with equal volumesof extracted DNA and 5 U of T4 DNA Ligase in a total volume of 20 [2l in1×T4 buffer (Roche Biochemicals). Ligation was allowed to proceed for 30min. at room temperature (RT). Of each ligation reaction, 3 μl were usedto transform E. coli XL-1 Blue as described above. Colonies were pickedand subjected to MiniPrep analysis. Following analytical restrictionenzyme digestion, appropriate clones were sequenced (Sequiserve,Munich). Correct clones were grown in 300 ml LB-Amp medium, and plasmidDNA was isolated using the Qiagen plasmid prep kit as described above.The corresponding nucleotide and amino acid sequences of this specific,illustrative construct are depicted in SEQ ID NOs: 3 and 4,respectively.

EXAMPLE 4 Expression and Purification of MOG×CD3 Fusion Protein

4.1. Stable Transfection of CHO Cells

CHO cells were plated at 3*10⁵/well in tissue culture 6-well plates andincubated at 37° C. overnight. 3 μg of DNA were pipetted in sterileEppendorf tubes, supplemented with 100 μl MEM-α medium (Gibco BRL) and10 μl SuperFect transfection reagent (Qiagen) and incubated for 10 min.at RT. 600 μl of MEM-α medium were added, and the reaction mixture wastransferred to CHO cells. Following a 2 h-incubation at 37° C., thesupernatant was aspirated, cells were washed once with PBS, and 2 mlMEM-α medium (10% FCS, HT-supplement 1:100) were added to each well.Transfection efficiency was determined to be 10% via standardβ-galactosidase control transfection. After 24 h at 37° C., transfectedcells were transferred to 10 ml cell culture bottles (Nunclone Δ, NalgeNunc International) and selected for expression of the dhfr vector viagrowth in non-supplemented MEM-α medium plus 10% dialysed FCS. Following2 passages of confluent cells at 1:5 splitting ratios, transfectantswere further selected by addition of 20 nM methotrexate (MTX) to theselection medium. Cells were passaged 3 times, whereafter MTXconcentration was increased to 100 nM. Following a further 3 passages,MTX was added to a final concentration of 500 nM.

4.2. Purification of Expressed MOG×CD3 Fusion Protein

Stably transfected CHO-cells were transferred to 500 ml roller-bottles(Nalge Nunc International) in 250 ml MEM-α, 500 nM MTX and 5% dialysedFCS. The following day, another volume of medium was added without FCSto obtain a final concentration of 2.5% FCS. Cells were grown for 1 daypost confluency. Cells were separated from the supernatant bycentrifugation at 4500 rpm, 30 min. in a Rotanta 46 centrifuge, andrecombinant protein was purified from cell culture supernatant in athree-step purification process including cation exchangechromatography, immobilized metal affinity chromatography (IMAC) and gelfiltration.

GradiFrac System (Pharmacia) was used for chromatography. All chemicalswere of research grade and purchased from Sigma (Deisenhofen) or Merck(Darmstadt).

Cation exchange was performed on a HiTrap SP Sepharose column(Pharmacia) that was equilibrated with buffer A1 (20 mM MES pH 5.5).Cell culture supernatant was diluted 2:1 with buffer Al and applied tothe column (10 ml) with a flow rate of 4 ml/min. Unbound sample waswashed out with buffer A1 and the bound protein was eluted with 100%buffer B1 (20 mM MES pH 5.5, 1M NaCl). Eluted protein fractions werepooled for further purification.

IMAC was performed, using a HisTrap column (Pharmacia) that was loadedwith NiSO4 according to the manufacturer's protocol. The column wasequilibrated with buffer A2 (20 mM NaPP pH 7.5, 0.4 M NaCl), and thesample was diluted 2:1 with buffer A2 to obtain a pH of 7. The samplewas applied to the column (2 ml) with a flow rate of 1 ml/min and thecolumn was washed with buffer A2 to remove unbound sample. Bound proteinwas eluted using a linear gradient of buffer B2 (20 mM NaPP pH 7.5, 0.4M NaCl, 0.5 M Imidazol) (0-100% buffer B2 in 10 column volumes). Elutedprotein fractions were pooled for further purification.

Gel filtration chromatography was performed on a Sephadex S200 HiPrepcolumn (Pharmacia) equilibrated with PBS (Gibco). Eluted protein samples(flow rate 1 ml/min) were subjected to SDS-Page and western blofting forMOG×CD3 detection. The column was previously calibrated for molecularweight determination (molecular weight marker kit, Sigma MW GF-200).

Protein concentrations were determined using protein assay dyeconcentrate (BioRad) and IgG (Biorad) as standard protein.

SDS-PAGE under non-reducing conditions was performed with precast 4-12%Bis-Tris gels (NOVEX). Sample preparation and application were accordingto the manufacturer's protocol. The molecular weight was determined withSeeBlue protein standard (NOVEX). The gel was stained with colloidalCoomassie (NOVEX protocol; FIG. 3A).

4.3. Western Blot

Fractions were analyzed by Western Blot and staining with anti-MOGantibody for the presence of MOG×CD3. Protein was blotted to reinforcednitrocellulose membrane (Optitran BA-S 83, Schleicher & Schuell) at 200mA for 1 h (blotting buffer: 48 mM Tris, 39 mM Glycin, 0.01% SDS).Recombinant fusion protein was detected with anti-MOG monoclonalantibody (8.18C5) at 5 μg/ml in PBS; bound anti-MOG ab was detected viaanti-mouse IgG antibody, AP-conjugated at 1:10000 in PBS (Sigma A-2429).The membrane was stained with BCIP/NBT (Sigma B-5655). (FIG. 3B)

The purity of the isolated protein was >95% as determined by SDS-PAGE.The molecule had an apparent mass of 45 kDa consistent with thepredicted size. The final yield of purified protein was ca. 2.4 mg/lcell culture supernatant. The final product ran as an approximately 45kDa protein under native conditions as determined by gel filtration inPBS. No higher molecular weight forms were detected, suggesting thatMOG×CD3 is a monomer (see FIG. 3B).

EXAMPLE 5 Expression and Purification of MOG-Fc Fusion Protein

5.1. CHO Cells were Transfected as Described in Example 4.

Stably transfected CHO-cells were transferred to 500 ml roller-bottles(Nalge Nunc International) in 250 ml MEM-α, 500 nM MTX and 5% dialysedFCS. The following day, another volume of medium was added without FCSto obtain a final concentration of 2.5% FCS. Cells were grown for 1 daypost confluency. Cells were separated from the supernatant bycentrifugation at 4500 rpm, 30 min. in a Rotanta 46 centrifuge, andrecombinant protein was purified using a 1-step purification procedurevia Protein A affinity chromatography (HiTrap Protein A column,Pharmacia) on the GradiFrac System (Pharmacia). Column was equilibratedwith 10 ml of buffer A (20 mM Tris pH 7.2), and 500 ml of cell culturesupernatant were passed through the column. Flow rate was 2 ml/ min.Bound Protein was eluted with 20 mM citrate, pH 3, using a lineargradient. Fusion protein yield amounted to 10 mg/l. Protein was >95%pure as assessed by Coomassie staining (FIG. 4A).

5.2. Protein Analysis of MOG-Fc

MOG-Fc fusion protein was analyzed via SDS-PAGE as described forMOG×CD3. Coomassie Brilliant Blue staining under reducing conditions(FIG. 4A) revealed a major band at 50 kDa, with a higher molecularweight lane at approximately 115 kDa. Western blotting under reducingand non-reducing conditions was performed for further analysis (FIG.4B); under reducing conditions (FIG. 4B, +DTT), MOG-Fc runs as a monomerof 50 kDa, which corresponds to its predicted size. The native proteinhas a molecular mass of approximately 115 kDa (FIG. 4B, −DTT) undernon-reducing conditions. This suggests that MOG-Fc is a disulfide-bridgelinked dimer in its native state, presumably cross-linked via twocysteine residues in the hinge-region of the human IgG1 part of theprotein.

EXAMPLE 6 Binding of Auto-Antigen Fusion Proteins to AutoreactiveB-Cells

6.1. Source and Isolation of Autoreactive B-Cells

Litzenburger et al. (1998) J. Exp. Med. 188 (1):169-180) generated atransgenic mouse strain with an anti-MOG heavy chain variable region,derived from the anti-MOG mAb 8.18-C5 “knocked in” for the germlne JHlocus. Such mice exclusively express the 8.18-C5 anti-MOG heavy chain,resulting in generation of approximately 30% MOG-reactivity in theB-cell pool, as assessed by binding to recombinant MOG. Wholelymphocytes from transgenic knock-in mice were prepared from spleen asdescribed elsewhere (Iglesias J. Exp. Med. 188 (1): 169-180). RestingB-lymphocytes (CD43-) were isolated from whole lymphocyte preparations.Cells were suspended in 5 ml PBS, 1% BSA, and 150 μl biotinylatedanti-CD43 ab (Pharmingen) were added. After 30 min. on ice, cells werewashed twice with PBS/1% BSA, and streptavidin-conjugated Dynabeads wereadded to obtain a mean 3.3 beads/cell. The mixture was incubated at 4°C. for 30 min. while rotating, whereafter bound cells were separatedmagnetically.

6.2. FACS-based Binding Assay of Auto-Antigen Fusion Protein toAutoreactive B-Cells

Autoreactive B-cells were washed twice with FACS-buffer (PBS, 1% FCS,0.05% NaN3). Cells were incubated with 50 μl fusion protein diluted to 1and 10 μg/ml in FACS-buffer, respectively for 1 h at 4° C. Bound MOG-Fcfusion protein was detected with goat-α IgG FITC/hu, Fc-specific (ICN67-217) at 1:50 (FIG. 6), while bound MOG×CD3 was detected withFITC-labeled a-HIS 6 antibody (Dianova; FIG. 7).

EXAMPLE 7 Binding of Auto-Antigen Fusion Protein to Auto-Antibody

7.1. Source of Auto-Antibodies

Hybridoma 8.18-C5 (Linington, MPI Neurobiology Martinsried) wascultivated in serum-free medium (Gibco). Cells were separated fromsupernatant by centrifugation, and mouse anti-MOG monoclonal IgG1antibodies were purified using a 1-step purification procedure viaProtein G affinity chromatography (HiTrap Protein G column, Pharmacia)on the GradiFrac System (Pharmacia). Column was equilibrated with 10 mlof buffer A (20 mM Tris pH 7.2), and 500 ml of cell culture supernatantwere passed through the column. Flow rate was 2 ml/min. Bound Proteinwas eluted with 20 mM citrate, pH 3, using a linear gradient. Antibodyyield amounted to 4.5 mg/l. Protein was >95% pure as assessed byCoomassie staining.

7.2. Sandwich-ELISA for Detection of MOG-Fc Fusion Protein

Isolated αMOG ab 8.18-C5 was used to detect purified MOG-Fc fusionprotein and to verify existence of 1) functional extracellular domain ofMOG protein and 2) Fc effector domain in the recombinant protein.MaxiSorp 96-well plates (Nalge Nunc International) were coated with aMOGat 5 μg/ ml overnight at 4° C. Plates were blocked with 1% BSA for 1 hat RT, washed with PBS/0.05% Tween 20. Plates were incubated withvarious dilutions of MOG-Fc fusion protein in PBS for 1 h at RT, andbound fusion protein was detected using a-human IgG1 ab, Fc-specific andAP-conjugated (Sigma A-9544) at 1:10,000. Alkalinephosphatase-conjugated antibody was stained with pNPP (Sigma N-2770) andquantitated on the SpectraFluor ELISA reader (Tecan).

7.3. Sandwich-ELISA for Detection of MOG×CD3 Fusion Protein

Isolated aMOG ab 8.18-C5 was used to detect purified MOG×CD3 fusionprotein and to verify existence of 1) functional extracellular domain ofMOG protein and 2) anti-CD3 effector domain in the recombinant protein.MaxiSorp 96-well plates (Nalge Nunc International) were coated with αMOGat 5 [2g/ml overnight at 4° C. Plates were blocked with 1 % BSA for 1 hat RT, washed with PBS/0.05% Tween 20. Plates were incubated withvarious dilutions of MOG×CD3 fusion protein in PBS for 1 h at RT, andbound fusion protein was detected using chicken polyclonal serum againstscFv anti-CD3 (Davids Biotechnology). Bound chicken Ab was detectedusing alkaline phosphatase (AP)-coupled donkey-a-chicken ab at 1:10000(Dianova 703-055-155). Alkaline phosphatase-conjugated antibody wasstained with pNPP (Sigma N-2770) and quantified on the SpectraFluorELISA reader (Tecan, FIG. 5B).

EXAMPLE 8 Binding of Auto-Antigen Fusion Proteins to Immune EffectorCells

8.1. Isolation of PBMCs

Buffy coats were diluted 1:2 in PBS and separated in Ficoll gradient ofdensity 1.077 (Seromed Cat.No. L 6115). Lymphocytes were separated andwashed twice with PBS. Erythrocytes were lysed with lysis buffer (8,29 gNH4Cl cell culture tested (Sigma A-0171), 1,0 g KHCO3 0,037 g EDTA, cellculture tested (Sigma E-6511);H₂O add. 1 L). Thrombocytes were separatedduring 20 min of centrifugation at 100×g. Remaining Lymphocytes weretransferred to cell culture bottles and stored at 37° C./ 5% CO2.

8.2. Isolation of CD3+ Cells

Human T-cell enrichment columns (R&D Systems Cat. No. HTCC-500/525) wereused for isolation of T-cells according to manufacturer's suggestions.

8.3. Binding Assay of MOG×CD3 to CD3+ PBMCs

To explore the binding of MOG×CD3 fusion protein to CD3+ cells, 200,000CD3+ cells were added to each well of a V-bottom microtiter plate(Greiner Labortechnik). Recombinant MOG×CD3 protein was added to obtainfinal concentrations of 0.032 up to 100 μg/ml in a total volume of 50 μlper well. Bound fusion protein was detected via binding of 8.18-C5diluted 1:1000 in FACS-buffer, whereafter bound mouse monoclonalantibody was stained with α-mouse IgG-FITC (Sigma F-6257) at 1:40dilution (FIG. 6).

EXAMPLE 9 Establishment of a Cytotoxicity Assay for MOG×CD3 and MOG-FcFusion Proteins

9.1. Establishment of Cell-Surface αMOG-positive Hybridoma Cell Line

8.18-C5 hybridoma cell line (Schluesener, J. Immunol. 139 (1987),4016-4021) was adapted to serum-free medium (Hybridoma SFM, Gibco).Cells were passaged 1:5 every third day, and cultured in 100% SFM for aperiod of 4-5 months. Thereafter, MOG-reactivity in the hybridoma poolwas assessed by FACS-analysis, using biotinylated MOG protein forstaining. Positive cells were identified and isolated individually in96-well plates by FACS-sorting. Clones were expanded for a period ofapproximately 2 weeks, and MOG-reactivity was checked again as describedabove. Anti-MOG positive clones were identified, and those showing thegreatest amount of MOG-reactivity were expanded and used as targets forin-vitro cytotoxicity assays. More than 90% of cells show cell-surfaceexpression of migG1 (FIG. 8A) and bound rMOG (FIG. 8B).

9.2. Selective Elimination of Autoreactive B-cells

A FACS-based cytotoxicity assay was performed. Effector cells (500000),8.18-C5 target cells and fusion protein were added in a total volume of200 μl RPMI/10% FCS to each well of a sterile round-bottom multititreplate (CoStar) and incubated overnight at 37° C. Target cells were addedto obtain E:T-ratios of 10:1, and MOG×CD3/-Fc fusion protein was addedto attain final concentrations of 0.1, 1 and 10 μg/ ml. Cells wereincubated for 16 h at 37° C., washed with FACS-buffer, and target cellswere labeled with anti-murin IgG1 antibody (Sigma 6257) at 1:50dilutions in FACS-buffer; incubation was performed at RT for 30 min.Dead cells were excluded by staining with propidium iodide, and cellswere analyzed with a FACSCalibur (Becton Dickinson). Dose-dependentcytotoxicity is shown for MOG×CD3 in FIG. 9A and for MOG-Fc in FIG. 9B.Specific toxicity is shown in FIG. 10, assay conditions were asdescribed above, unspecific toxicity was defined by cytotoxicitymeasured in the absence of protein. Spontaneous lysis was determined byseparately incubated target and effector cells in the absence ofprotein, the cells were mixed prior to FACS analysis. The negativevalues reflect slight proliferation during the 16 h incubation.

EXAMPLE 10 Inhibition of MOG×CD3 Cytotoxicity by Recombinant MOG (rMOG)

The specificity of MOG×CD3 based cytotoxicity was tested by addition ofsoluble recombinant MOG. A FACS-based cytotoxicity assay was performedas described in example 9, using MOG×CD3 at a concentration of 0.1μg/ml. Recombinant, biotinylated MOG (rMOG) was added to a finalconcentration of 20 μg/ml and the cytotoxicity assay performed for 16 h.As shown in FIG. 11, cytotoxic activity can be inhibited by the additionof rMOG.

EXAMPLE 11 MOG×CD3 Binds to 8.18-C5 Target Cells

Binding of MOG×CD3 to B cell carrying membrane Ig receptor specific forthe MOG protein was tested by a FACS-based binding assay. 8.18-C5 targetcells were incubated with purified MOG×CD3 fusion protein at variousconcentrations for 1 h at 4° C. Cells were washed twice withFACS-buffer, and bound fusion protein was detected through itsC-terminal HIS-tag using FITC-labeled anti-HIS antibody (Dianova). Cellswere analyzed by FACS scanning and mean fluorescence scores werecalculated and are shown in FIG. 12. MOG×CD3 efficiently bound atconcentrations below 5 μg/ml.

EXAMPLE 12 Selective Elimination, of Autoreactive B-Cells at DifferentEffector-to-Target Cell Ratios (E:T)

The dependency of MOG×CD3 cytotoxicity on the availability of effectorcells was determined by a FACS-based cytotoxicity assay as described inexample 9. Target cells were seeded at a constant density of 50000cells/well, while the CD3+human effector cell concentration was variedin order to obtain the desired E:T ratio. FIG. 13 shows thecorresponding specific toxicity, demonstrating that MOG×CD3 cytotoxicityis dependent on effector cell number.

EXAMPLE 13 Binding Specificity of Auto-Antigen Fusion Proteins toAutoreactive B-Cells

13.1. Specific Binding of MOG-Fc Fusion Protein to IqM+ B-cells Derivedfrom Splenocytes from Anti-MOG Transgenic Mice

Binding of MOG-Fc as described in Example 3 and FIG. 4 to MOG-reactive Bcells was investigated using splenocytes from anti-MOG transgenic mice(FIG. 14A, lower panel). In these mice, the endogenous heavy chain-Jregion has been replaced by the rearranged 8.18-C5 anti-MOG VDJ segmentusing knock-in technology (Litzenburger, J. Exp. Med. 188 (1998),169-180). As a result, almost all B cells of transgenic mice express theMOG-specific heavy chain in combination with endogenous light chains.

Whole lymphocytes from transgenic knock-in mice were prepared fromspleen as described elsewhere (Litzenburger, J. Exp. Med. 188 (1998),169-180). Cells were incubated with MOG-Fc fusion protein for 20 min onice, and bound MOG-Fc was detected with goat-anti-human IgG FITCantibody (ICN 67-217). It was shown in FACS analysis that >90% of allIgM-positive splenocytes from the anti-MOG transgenic mice (FIG. 14A,lower panel) but not of control littermate mice (FIG. 14A, upper panel)did bind recombinant MOG-Fc. Two populations of B-cells were observed:Approximately one third of IgM-positive B cells in anti-MOG transgenicmice bound MOG-Fc with high intensity while two thirds bound MOG-Fc withlow intensity (FIG. 14A, lower panel). This binding pattern wasreminiscent of the one reported using biotinylated, bacteriallyproduced, recombinant rat MOG (rMOG) (Litzenburger, J. Exp. Med. 188(1998), 169-180 and Litzenburger, J. Immunol. 165, (2000) 5360-5366). Inthis report, however, two thirds of the B cells derived from anti-MOGtransgenic mice did not bind rMOG at all or only poorly, and only onethird of B cells bound rMOG with a broad range of intensities. Suchdifferences are likely due to an intrinsically higher affinity of theseB cells to MOG-Fc which, in contrast to the bacterially generated rMOG,is dimeric, properly folded, N-glycosylated and has not undergonebiotinylation.

13.2. Selective Binding of MOG-Fc Fusion protein to Wildtype MurineSplenocytes

Binding of MOG-Fc (as described in Example 3 and FIG. 4) to wildtypesplenocytes was also investigated. Single-cell suspensions of wildtypesplenocytes were prepared. Cells were incubated with 50 μg/ml MOG-Fcprotein or PBS.

In order to allow binding to low-affinity Fc receptors, incubation withMOG-Fc was performed with 10-times the concentration of protein as withsplenocytes from anti-MOG transgenic mice (Litzenburger, J. Exp. Med.188 (1998), 169-180) and for a period of 45 minutes at room temperaturecompared to 20 minutes on ice. The subsequent incubation with anti-Mac1,anti-CD5 or biotinylated 8.18-C5 antibodies was performed on ice for 20min. As shown in FIG. 14B, MOG-Fc did bind to the Mac1^(high) (CD11b, BDPharmingen) positive population, indicating a preferential binding tomacrophages and myeloid CD8⁺ dendritic cells (DCs). The upper panels ofFIG. 14B and C show incubations on ice alone without prior incubationwith MOG-Fc at room temperature. No binding of MOG-Fc to CD5⁺ T cellscould be detected emphasizing the selectivity for FcγR⁺ cells.

13.3. Specific Binding of MOG-Fc Fusion Protein to Murine MacrophageCell Line

The murine monocyte/macrophage cell line p388.D1 also bound MOG-Fc (asdescribed in Example 3 and FIG. 4) as shown by FACS detection of MOG-Fcvia FITC-labeled anti-human Fcγ antibody (FIG. 1D). Mean fluorescenceintensity (MFI) values observed for MOG-Fc were similar to thoseobtained with an isotype control IgG1 (Raum, Cancer Immunol Immunother.50, (2001), 141-150) (data not shown).

EXAMPLE 14 Specificity of MOG-Fc Mediated Cytotoxicity

The specificity of cell lysis mediated by MOG-Fc (as described inExample 3 and FIG. 4) was investigated by using both unrelated humanIgG1 (Raum, Cancer Immunol Immunother. 50, (2001), 141-150) and a mouseB cell line expressing unrelated IgG on its surface (TIB-208).

14.1. Effect of Non-Specific Human IqG1

8.18-C5 target cells were incubated with human PBMCs and 10 μg/ml MOG-Fcprotein (as described in Example 3 and FIG. 4) in RPMI with 10% FCS.Incubation was performed for 16 h at 37° C./5% CO₂. After incubation,target cells were detected with FITC-labeled anti-murine IgG1 antibody(mlgG, Pharmingen) and analyzed for viability through propidium iodide(PI) staining. The EpCAM antigen-specific recombinant human control IgG1(Raum, Cancer Immunol Immunother. 50, (2001), 141-150) can bind to humaneffector cells via its Fc part but not to MOG-specific immunoglobulin onhybridoma cells. This isotype control did not lead to significantredirected lysis of 8.18-C5 cells (FIG. 15A).

14.2. Effect of an Unrelated Murine Cell Line Expressing Cell SurfaceIgG1

Mouse B cell line TIB-208 is expressing cell surface IgG1 of non-MOGspecificity and was incubated with human PBMCs and 10 μg/ml MOG-Fcprotein (as described in Example 3 and FIG. 4) in RPMI with 10% FCS.Incubation was performed for 16 h at 37° C. and 5% CO₂. The mouse B cellline TIB-208 expressing cell-surface IgG of non-MOG specificity was notsensitive to MOG-Fcmediated cell lysis (FIG. 15B).

EXAMPLE 15 MOG-Fc Mediated Depletion of Splenocytes of Anti-MOGTransgenic Mice ex Vivo

To investigate the cytotoxic potential of MOG-Fc (as described inExample 3 and FIG. 4) on MOG-specific B cells in vivo, the fusionprotein was tested in anti-MOG transgenic mice. Firstly, anti-MOG micehave an extremely high titer of circulating MOG-reactive antibodies(Litzenburger, J. Exp. Med., 188, (1998)169-180) and secondly, almostall B cells in these mice do show MOG reactivity. MOG-Fc had the abilityto deplete primary anti-MOG-positive B cells from anti-MOG transgenicmice ex vivo and in vivo. Elimination of MOG-reactive B cells ex vivowas efficient (on the order of 70% within 16 hours), while in vivo itwas lower but still significant among the population of highlyMOG-reactive B cells. MOG-reactive B cells are constantly replenished inthe periphery by the bone marrow on the order of 10⁷ cells/day. Incontrast, the frequency of autoreactive B cells in human autoimmunedisease is extremely low (Link, J. Clin. Invest., 87, (1991), 2191-2195and Nishifuji, J. Invest. Dermatol., 114, (2000), 88-94). Therefore, theanti-MOG transgenic mouse model represented the most difficult situationand highest possible hurdle to test the in-vivo efficacy of a specific Bcell-eliminating protein such as MOG-Fc.

In order to assess whether MOG-Fc (as described in Example 3 and FIG. 4)can also eliminate normal B cells expressing MOG-reactive cell-surfaceimmunoglobulin, splenocytes from anti-MOG transgenic mice were isolatedand incubated with 10 μg/ml of MOG-Fc fusion protein for 16 h at 37° C.and 5% CO₂ in DMEM with 10% FCS in 5 ml cell culture polypropylene vials(Becton-Dickinson, Pharmingen) at a density of 4×10⁶ cells/ml.Lymphocyte analysis was carried out by FACS using biotinylatedrecombinant MOG protein and antibodies against IgMa, IgD and CD19 (allBecton-Dickinson, Pharmingen). All tests were carried out in triplicate.The endogenous Fcy receptor-bearing cells served as effectors. In FACSanalysis, a highly significant depletion of B cells positive for IgMa,IgD and CD19 was observed (FIG. 16). The average degree of B celldepletion was on the order of 70% and was comparable for all threeB-cell markers.

EXAMPLE 16 In Vivo Depletion of Anti-MOG Specific B-Cells in TransgenicMice

The cytotoxic efficacy of MOG-Fc (as described in Example 3 and FIG. 4)was tested in vivo using the anti-MOG transgenic mouse. This mousestrain carries the anti-MOG heavy chain variable region, derived fromthe anti-MOG monoclonal antibody 8.18-C5 “knocked in” for the germlineJH locus (Litzenburger, J. Exp. Med. 188 (1998), 169-180).

Female anti-MOG transgenic mice were treated twice with 100 μg ofrecombinant MOG-Fc fusion protein in 500 μl PBS through i.p. injection(n=5) on day 1 and 3. Blood was collected each day after treatment, PBLswere prepared, and lymphocytes were analyzed via FACS (FACSCalibur,Becton-Dickinson) analysis one day post treatment. MOG⁺ B cells werequantitated and normalized as percentage of total B220⁺ B cells detectedin the lymphocyte gate. MOG-Fc was efficient in depleting IgM-positive Bcells in the fraction that strongly reacted with MOG (FIG. 17A, upperpanel). MOG-Fc was still bound to B cells 24 h post-treatment (FIG. 17B)as detected by staining of isolated B220-positive blood lymphocytes withanti-human Fcy specific antibody.

The percentage of highly MOG expressing B-cells decreased withstatistical significance from 45% in control (untreated mice) to lessthan 30% on day 4 after the first treatment (FIG. 17B). The number ofB220⁺ B cells was decreased to the same extent as the number ofIgM-positive B cells (data not shown), suggesting that the reduction ofB cells measured by flow cytometry was due to a depletion of cells andnot to a down-modulation of the B-cell receptor expression.

EXAMPLE 17 Depletion of Autoreactive B Cells and Reduction ofMOG-Specific IgG in Wildtype Mice After Cellular Transfer

17.1 In Vivo Depletion of Anti-MOG Reactive B Cells in Wildtype MiceAfter Cellular Transfer

In order to study the depleting potential of MOG-Fc on a limited numberof B-cells derived from anti-MOG transgenic mice in vivo, 1.5×10⁷ Bcells derived from anti-MOG transgenic mice with BL/6 background weretransferred intraveniously into wt BL/6.

Wild-type mice transferred with a limited number of autoreactive B-cellscan be used as a model system reflecting the situation inB-cell-mediated autoimmunity which is characterized by the presence of alimited number of autoreactive B-cells versus a high background ofendogenous normal B lymphocytes. Thus, this system is very close to thereal-life situation and confirmes the data on depletion of MOG-reactiveB cells obtained in the anti-MOG transgenic mice.

Mice were divided into 3 groups (N=5 per group). Group A was treatedwith MOG-Fc (100 ug each treatment) intraperitoneally 1, 2 and 3 dayspost-transfer. Control groups were treated with human IgG1 isotypecontrol (Raum, Cancer Immunol Immunother. 50, (2001), 141-150, group B)and PBS (group C). For this purpose splenocytes derived from anti-MOGtransgenic mice with BL/6 background were prepared as single-cellsuspension and stimulated with LPS as described (Litzenburger, J. Exp.Med. 188 (1998), 169-180). Three days post-stimulation at 37° C./ 5%CO₂, resting B-cells (CD43-) were isolated (Litzenburger, J. Exp. Med.188 (1998), 169-180) and 1.5×10⁷ cells were transferred intravenouslyinto wildtype BL/6 mice on the same day. Mice were treated as describedabove on days 1,2 and 3 following transfer. 24 h and 72 h after the lasttreatment, peripheral blood was collected by tail bleeding and analyzedfor anti-MOG reactive B cells via FACS staining (FACS Calibur,Becton-Dickinson) with recombinant MOG-Fc (as described in Example 3 andFIG. 4) followed by detection with PE-conjugated anti-human Fc antibody(ICN).

One day post-treatment with MOG-Fc depletion of MOG-reactive B cells(FIG. 18A) was observed. This effect was more prominent three dayspost-treatment showing a depletion af autoreactive B-cells in the orderof 70% (FIG. 18B).

17.2. Reduction of MOG-specific IgG in Wildtype Mice After CellularTransfer

1.5×10⁷ B cells derived from anti-MOG transgenic mice with BL/6background were transferred intraveniously into wt BL/6 as described inExample 17.1. Mice were divided into 3 groups (N=5 per group) andtreated with MOG-Fc (100 ug each treatment) intraperitoneally 1, 2 and 3days post-transfer (A), human IgG1 isotype control (Raum, Cancer ImmunolImmunother. 50, (2001), 141-150, group B) and PBS (group C). Peripheralblood was collected by tail bleeding and sera from day 1 and 3post-treatment were analyzed for anti-MOG specific IgG titers by ELISA.ELISA plates were coated with recombinant MOG (10 μg/ml), serum samplescollected 24 h post-treatment were applied at 1:100 dilution and serumsamples collected at 72 h post-treatment were applied at 1:1000 dilutionin PBS. Bound MOG-reactive IgG was detected with biotinylatedrat-anti-mouse IgG antibody (Jackson Laboratories). Streptavidin-AP andpNPP (Sigma N-2770) were used for detection and OD values werequantified on a Dynatech MR4000 ELISA reader. MOG-Fc had the ability toreduce the number of circulating, MOG-reactive IgG after 24 h (FIG. 19A)and 72 h (FIG. 19B), presumably via a combination of direct adsorptionand neutralization of MOG-reactive immunoglobulin.

Both effects described in Example 17.1. and 17.2. are of hightherapeutic value, since immunoadsorption of autoreactive immunoglobulinleads to a short-term effect through depletion of pathogenic antibodies,while elimination of autoreactive B cells depletes the pathogeniccellular reservoir.

EXAMPLE 18 Generation and Use of a De-Immunized MOG-Fc Construct

18.1 The generation and use of MOG-Fc construct for the depletion ofautoreactive B-cells have been described herein above. Here, anillustrative example for the generation and use of de-immunized(poly)peptide construct in accordance with this invention is given.

18.2 Selection of an Immunodominant Epitope of MOG

In the chosen model system (SJL-J mouse), immunization with MOG inducesa T-cell response against 2 main regions: sequence 1-22 and 92-106 (M36)of the MOG extracellular domain (Gardinier, 1992, J. Neurosci. Res., 33,177-187). While clones against 1-22 are incapable of inducing disease,clones versus M36 may be encephalitogenic and may induce EAE upontransfer into recipient animals (Amor, 1994, J. Immunol. 153,4349-4356). Therefore, the M36 region was analyzed for amino acidresidues critical for the contact with the T-cell receptor (TCR) via AlaScanning. Corresponding, modified M36 regions of the human M36 are shownin SEQ ID NOs: 12 to 26. SEQ ID NO: 10 depicts wildtype M36.

Proliferation response of M36-specific T-cell-line (kindly provided byA. Iglesias, MPI Neurobiology Martinsried) to Ala-substituted peptideswas tested via standard 3H-thymidine incorporation assay (see FIG. 20).Briefly, T-cells were cultured for a period of 14 days postre-stimulation with wildtype-peptide (wt). For assessment ofproliferation, T-cells were transferred to restimulation medium (DMEMcomplete, 5% FCS). Wild-type SJL/J splenocytes were prepared by standardprotocol (Litzenburger, 1998, J. Exp. Med. 188, 169-180) and irradiatedwith 4000 rad for use as APCs. The assay was set up in flat-bottom96-well tissue culture plates. APCs and T cells were seeded at 10:1(106:105 per well) and incubated with 10 μg/ ml of peptide at 37° C./ 5%CO₂. After 48 h, the cells were pulsed with ³H-thymidine (1 μCi/ ml).Thymidine incorporation was investigated 16 h later. T-cellproliferation was not induced by peptides SEQ ID NO: 16, 19, 21, 23 and24 (FIG. 20). Specific proliferation of M36 T cell line was investigatedin a dose-response experiment (FIG. 21). Assay was performed asdescribed for FIG. 20 with varying concentrations of M36 wildtypepeptide (SEQ ID NO: 10).

Thymidine incorporation assay was performed to investigate the abilityof the mutated peptides to function as T-cell antagonists in a peptidecompetition assay, thus excluding interactions solely mediated throughdecreased binding to MHC II (De Magistris et al., 1992, Cell, 68,625-634; Windhagen et al., 1995, Immunity, 2, 373-380). APCs werepre-pulsed with 10 μg/ ml of wt M36 SEQ ID NO: 10 peptide for 2 h at 37°C./5% CO₂ (first stimulation). Cells were then washed with restimulationmedium and incubated with 10 μg/ ml of the substituted M36 variants(second stimulation). Proliferation was assessed as described above.Peptides substituted at positions 10, 12 and 13 (SEQ ID NO: 21, 23, 24)inhibited proliferation induced by human wildtype peptide (FIG. 22,hu/10, hu/12, hu/13).

18.3 Cloning, Expression and Purification of a De-Immunized ConstructExemplifying the Invention

Cloning of Mutated MOG-Fc Construct (eMOG-Fc)

Site-directed mutagenesis was performed by standard protocol. Briefly,primers were synthesized covering the 5′ and 3′ terminal sequence (5′:A, 3′: D) of the region encoding the MOG extracellular domain. Formutagenesis, primers were chosen to include the desired nucleotideexchanges flanked by 15 to 20 bases to both the 3′ and 5′ end (forwardprimer (5′): C, backward primer (3′): B). Primers A and B were used inPCR to generate fragment 1, primers C and D generated fragment II, eachusing MOG-Fc as a template. Following gel extraction, fragments I and IIwere used as templates to synthesize eMOG-Fc with primers. The resultingconstruct (eMOG-Fc) is depicted in SEQ ID NOs: 27 and 28, whereby SEQ IDNO: 27 corresponds to the coding nucleic acid molecule (DNA) and SEQ IDNO: 28 relates to the expressed (poly)peptide.

Expression and Purification of eMOG-Fc Fusion Protein

Stable Transfection of CHO Cells

CHO cells were plated at 3*10ˆ5/ well in tissue culture 6-well platesand incubated at 37° C. overnight. 3 ug of DNA were pipetted in sterileEppendorf tubes, supplemented with 100 ul MEM-α medium (Gibco BRL) and10 ul SuperFect transfection reagent (Qiagen) and incubated for 10 min.at RT. 600 ul of MEM-α medium were added, and the reaction mixture wastransferred to CHO cells. Following a 2h-incubation at 37° C., thesupernatant was aspirated, cells were washed once with PBS, and 2 mlMEM-α medium (10% FCS, HT-supplement 1:100) were added to each well.Transfection efficiency was determined to be 10% via standardβ-galactosidase control transfection. After 24 h at 37° C., transfectedcells were transferred to 10 ml cell culture bottles and selected forexpression of the dhfr vector via growth in non-supplemented MEM-αmedium plus 10% dialysed FCS. Following 2 passages of confluent cells at1:5 splitting ratios, transfectants were further selected by addi-tionof 20 nM MTX to the selection medium. Cells were passaged 3 times,whereafter MTX concentration was increased to 100 nM. Following afurther 3 passages, MTX was added to a final concentration of 500 nM.

Purification of Expressed eMOG-Fc Fusion Protein

Stably transfected CHO-cells were transferred to 500 ml roller-bottlesin 250 ml MEM-α, 500 nM MTX and 5% dialysed FCS. The following day,another volume of medium was added without FCS to obtain a finalconcentration of 2.5% FCS. Cells were grown for 1 day post confluency.Cells were separated from the supernatant by centrifugation at 4500 rpm,30 min. in a Rotanta 46 centrifuge, and recombinant proteih was purifiedusing a 1-step purification procedure via Protein A affinitychromatography (HiTrap Protein A column, Pharmacia) on the GradiFracSystem (Pharmacia). Column was equilibrated with 10 ml of buffer A (20mM Tris pH 7.2), and 500 ml of cell culture supernatant were passedthrough the column. Flow rate was 2 ml/ min. Bound protein was elutedwith 20 mM citrate, pH 3, using a linear gradient. Fusion protein yieldamounted to 10 mg/l. Protein was >95% pure as assessed by Coomassiestaining.

Selective Binding of eMOG-Fc to Autoreactive B Cells.

Whole lymphocytes from transgenic knock-in mice were prepared fromspleen as described elsewhere (Litzenburger, 1998, J. Exp. Med. 188,169-180). Cells were incubated with fusion protein, and bound eMOG-Fcwas detected with goat-anti-human IgG FITC antibody (ICN 67-217). Thesubsequent incubation with anti-IgM, anti-B220, and anti-CD5 Abs wasperformed on ice for 20 min.

Fusion protein was bound selectively by transgenic B-cells with MOGreactivity, but not by wildtype B-cells of littermate controls (FIG.23).

Ex-Vivo Elimination of B Cells by eMOG-Fc.

Splenocytes from TH mice (SJL/J background) were prepared. Single-cellsuspensions were incubated with eMOG-Fc (10 μg/ml) for 16 h at 37° C./5%CO₂ in DMEM/10% FCS in 5 ml cell culture polypropylene vials(Becton-Dickinson) at a density of 4×10⁶ cells/ml. Lymphocyte analysiswas carried out by FACS using antibodies against CD19 (BD Pharmingen).All tests were carried out in triplicate. B-cell numbers in e-MOG-Fctreated cultures were reduced from >12% of total live lymphocytes incontrols to <8% in e-MOG-Fc treated cultures (FIG. 24).

Strategies aimed at specifically targeting autoreactive T cells fordown-modulation with peptides encompassing predicted immunodominantT-cell epitopes in man have been pursuit in clinical phase I studies(Steinman, J. Ex. Med., 2001). These studies had to be halted due tonegative side effects, including evidence of exacerbation of disease.The format of inventive polypeptides described herein overcomes thechallenge posed by pathogenic T-cell epitopes in the following manner:The inventive construct, exemplified by eMOG-Fc was developed to excludeany T-cell activation induced by the remaining T-cell epitopes in theauto-antigen. This was achieved by identification of the immunodominantepitope and analysis of the fine specificity of T cells reactive againstthis epitope. Having identified critical T-cell receptor contact and/orMHC class II binding residues, the very same mutations into the wholeauto-antigen×effector domain protein were incorporated, as is shown inthe example described here. Identified alanine-mutated peptidesinhibited proliferation of a specific T-cell line. Mutated proteinsretained their binding activity to specific B-cell receptors (FIG. 23).Ex-vivo analysis showed that these proteins can ablate autoreactiveB-cells from transgenic animals using endogenous effectors (FIG. 24).Thus, the polypeptides of the invention provide for novel, inventiveformats which are useful for specific depletion of B-cells in theabsence of undesired T-cell activation.

EXAMPLE 19 Deimmunization of Human Acetylcholine Receptor (AchR) Linkedto an Effector Domain for Treatment of Myasthenia gravis

The extracellular domain from amino acids 1-210 of the human AchR alphachain was combined with an immunoglobulin Fc part for the recruitment ofimmune effector cells.

Construction/Purification/Characterization of AchR-Fc

Expression of the construct of interest is driven by the promotor of thehuman elongation factor alpha (Kufer; PNAS 92 (1995): 7021). Thispromotor is known to be very efficient in virtually all eukaryoticcells, thereby making this expression system a powerful tool for highprotein expression without limitations regarding the selected eukaryotichost cell line. A versatile multiple cloning site (MCS) facilitates thecloning of the construct. The expression of the construct of interest islinked to the expression of the selection marker dihydrofolate reductase(DHFR) via the internal ribosomal entry site (IRES). This arrangementassures that almost all stably transfected cells will express theconstruct, as both genes depend on the promotor of EFα. A strongpolyadenylation signal for both genes is provided by the SV40polyadenylation site, and the pUC18 backbone of the plasmid provides awell-characterized plasmid backbone with ampicillin resistance forbacterial selection.

Construction of Auto-Antigen—Fc Fusion Protein: AchR-Fc

19.1. Isolation of RNA from HD69-Transfected CHO Cells and cDNASynthesis.

Total RNA was isolated from HD69-transfected CHO cells (WO9846645) usingthe Qiagen RNEasy RNA Extraction Kit according to the manufacturerssuggestions. RNA was dissolved in H2O and stored at −80° C.Complementary DNA synthesis was performed: briefly, 2 μg total RNA wereadded to 0.5 μg Oligo-dT primer in a total reaction volume of 12 μl. Thereaction mixture was incubated at 70° C. for 10 min. Then, 4 μl 5×FirstStrand Buffer (Gibco BRL), 2 μl 0.1M DTT and 1 μl 10 mM dNTPs wereadded. Incubation was performed at 42° C. for 2 min, after which 200 Uof Superscript II Reverse Transcriptase (RT) (Gibco BRL) were added. Thereaction mixture was incubated for 50 min. at 42° C. Then, RT wasinactivated due to a 15 min incubation step at 70° C. Isolated cDNA wasstored at −20° C.

19.2. Amplification of IgG1-Coding cDNA Fragments

In order to obtain cDNA coding for the Fc-domain of the human IgG1antibody backbone, primers were designed to amplify the 699 bases codingfor the Fc part of the human IgG1 backbone. Amplification was performedaccording to standard PCR protocols. Briefly, 50 pmol/ each ofappropriate primer, 1 μl dNTPs 10 mM, 4 μl cDNA, 5 μl Pfu-buffer5×(Stratagene) and 5 U Pfu-Polymerase (Stratagene) were added to a finalvolume of 50 μl in H₂O.

DNA was recovered from PCR reaction mixture according to themanufacturer's suggestions (Boehringer High Pure PCR ProductPurification Kit, cat. no. 1 732 676). Blunt-ended PCR productsgenerated by Pfu DNA polymerase were ligated into pCR-script vector(Stratagene #211188) according to manufacturer's protocol (Stratagene).Plasmids were transformed into competent E. coli strain XL-1 Blue using4 μl of ligation product added to 50 μl of E. coli. The mixture wasincubated on ice for 10 min., 1 min. at 42° C., and then again on icefor 2 min. Thereafter, 150 μl LB-medium were added and expression ofampicillin resistance genes was induced due to 45 min at 37° C. whileshaking. Reaction mixtures were plated on LB-Amp Agarose plates (50 μgampicillin/ml) and incubated at 37° C. for 16 h. Colonies were pickedand grown in LB-Amp medium (100 μg/ ml) for 8-12 h. Bacteria were spundown, and plasmid DNA was isolated according to manufacturer'ssuggestions (Plasmid Mini-Kit, Qiagen). DNA was subjected to restrictionenzyme analysis, and suitable clones were sequenced (SequiServe,Munich). Correct clones were grown in 300 ml LB-Amp medium, and plasmidDNA was isolated according to manufacturer's instructions (PLasmid MaxiKit, Qiagen).

19.3. Construction of AchR-Fc Fusion Protein

In order to obtain the desired construct, expression vector CD19×CD3pEF-dhfr was subjected to restriction with EcoRI and Sall, leading toremoval of the fragment coding for CD1 9×CD3. The remaining linearizedvector was gel-extracted (gel extraction kit, Qiagen). AchR α-chaindomain corresponding to aa 1-210 (sequence published in Swiss-PROTdatabase, accession number P02708) was amplified in PCR by standardmethods. The Fc immunoglobulin part was amplified as described inexample 19.2. AchR fragment at the 5′ end was combined with the Fcfragment at the 3′ end of the AchR-Fc construct and inserted into theexpression vector. E. coli XL-1 Blue were transformed and colonies werepicked and subjected to MiniPrep analysis. Following analyticalrestriction enzyme digestion, appropriate clones were sequenced(Sequiserve, Munich). Correct clones were grown in 300 ml LB-Amp medium,and plasmid DNA was isolated using the Qiagen plasmid prep kit asdescribed above.

EXAMPLE 20 Expression and Purification of the Inventive, IllustrativeFusion Protein AchR-Fc

Stable Transfection of CHO Cells

CHO cells were plated at 3*10⁵/well in tissue culture 6-well plates andincubated at 37° C. overnight. 3 μg of DNA were pipetted in sterileEppendorf tubes, supplemented with 100 μl MEM-α medium (Gibco BRL) and10 μl SuperFect transfection reagent (Qiagen) and incubated for 10 min.at RT. 600 μl of MEM-α medium were adqed, and the reaction mixture wastransferred to CHO cells. Following a 2 h-incubation at 37° C., thesupernatant was aspirated, cells were washed once with PBS, and 2 mlMEM-a medium (10% FCS, HT-supplement 1:100) were added to each well.Transfection efficiency was determined to be 10% via standardβ-galactosidase control transfection. After 24 h at 37° C., transfectedcells were transferred to 10 ml cell culture bottles (Nunclone Δ, NalgeNunc International) and selected for expression of the dhfr vector viagrowth in non-supplemented MEM-α medium plus 10% dialysed FCS. Following2 passages of confluent cells at 1:5 splitting ratios, transfectantswere further selected by addition of 20 nM methotrexate (MTX) to theselection medium. Cells were passaged 3 times, whereafter MTXconcentration was increased to 100 nM. Following a further 3 passages,MTX was added to a final concentration of 500 nM.

Stably transfected CHO-cells were transferred to 500 ml roller-bottles(Nalge Nunc International) in 250 ml MEM-α, 500 nM MTX and 5% dialysedFCS. The following day, another volume of medium was added without FCSto obtain a final concentration of 2.5% FCS. Cells were grown for 1 daypost confluency. Cells were separated from the supernatant bycentrifugation at 4500 rpm, 30 min. in a Rotanta 46 centrifuge, andrecombinant protein was purified using a 1-step purification procedurevia Protein A affinity chromatography (HiTrap Protein A column,Pharmacia) on the GradiFrac System (Pharmacia). Column was equilibratedwith 10 ml of buffer A (20 mM Tris pH 7.2), and 500 ml of cell culturesupernatant were passed through the column. Flow rate was 2 ml/min.Bound Protein was eluted with 20 mM citrate, pH 3, using a lineargradient. AchR-Fc fusion protein was analyzed by SDS-PAGE.

EXAMPLE 21 Binding of Auto-Antigen Fusion Protein to Auto-Antibody

21.1. Source of Auto-Antibodies

Anti-AchR hybridoma cells (Fosteri, 2000 FEBS Left., 481, 27-30) werecultivated in serum-free medium (Gibco). Cells were separated fromsupernatant by centrifugation, and mouse anti-AchR monoclonal antibodieswere purified using a 1-step purification procedure via Protein Gaffinity chromatography (HiTrap Protein G column, Pharmacia) on theGradiFrac System (Pharmacia). Column was equilibrated with 10 ml ofbuffer A (20 mM Tris pH 7.2), and 500 ml of cell culture supernatantwere passed through the column. Flow rate was 2 ml/min. Bound proteinwas eluted with 20 mM citrate, pH 3, using a linear gradient.

21.2. Sandwich-ELISA for Detection of AchR-Fc Fusion Protein

AchR specific antibodies were used to detect purified AchR-Fc fusionprotein and to verify existence of 1) functional extracellular domain ofAchR protein and 2) Fc effector domain in the recombinant protein.MaxiSorp 96-well plates (Nalge Nunc International) were coated withanti-AchR overnight at 4° C. Plates were blocked with 1% BSA for 1 h atRT, washed with PBS/0.05% Tween 20. Plates were incubated with variousdilutions of AchR-Fc fusion protein in PBS for 1 h at RT, and boundfusion protein was detected using α-human IgG1 ab, Fc-specific andAP-conjugated (Sigma A-9544) at 1: 10,000. Alkalinephosphatase-conjugated antibody was stained with pNPP (Sigma N-2770) andquantitated on the SpectraFluor ELISA reader (Tecan).

EXAMPLE 22 Binding of AchR-Fc Fusion Proteins to Immune Effector Cells

Isolation of PBMCs

Buffy coats were diluted 1:2 in PBS and separated in Ficoll gradient ofdensity 1.077 (Seromed Cat.No. L 6115). Lymphocytes were separated andwashed twice with PBS. Erythrocytes were lysed with lysis buffer (8,29 gNH4Cl cell culture tested (Sigma A-0171), 1,0 g KHCO3 0,037g EDTA, cellculture tested (Sigma E-6511);H2O add. 1L). Thrombocytes were separatedduring 20 min of centrifugation at 100×g. Remaining Lymphocytes weretransferred to cell culture bottles and stored at 37° C./5% CO2.Purified PBMC were incubated with the AchR-Fc fusion protein. AchR-Fcfusion protein was bound to Fcγ receptor positive cells via its Fc partas shown by FACS staining with anti-AchR antibodies.

EXAMPLE 23 Cytotoxicity Assay for AchR-Fc Fusion Proteins

23.1. Establishment of Cell-Surface αAchR-Positive Hybridoma Cell Line

Hybridoma cell line (Fostieri, 2000, FEBS Lett. 481, 27-30) was adaptedto serum-free medium (Hybridoma SFM, Gibco). Cells were passaged 1:5every third day, and cultured in 100% SFM for a period of 4-5 months.Thereafter, AchR-reactivity in the hybridoma pool was assessed byFACS-analysis, using biotinylated AchR protein for staining. Positivecells were identified and isolated individually in 96-well plates byFACS-sorting. Clones were expanded for a period of approximately 2weeks. Anti-AchR positive clones were identified and used as targets forin-vitro cytotoxicity assays.

23.2. Selective Elimination of Autoreactive B-Cells

A FACS-based cytotoxicity assay was performed. Effector cells (500000),hybridoma target cells (Fostieri, 2000, FEBS Left., 481, 27-30) andfusion protein were added in a total volume of 200 μl RPMI/10% FCS toeach well of a sterile round-bottom multititre plate (CoStar) andincubated overnight at 37° C. Target cells were added to obtainE:T-ratios of 10:1, and AchR-Fc fusion protein was added to attain finalconcentrations of 0.1, 1 and 10 μg/ ml. Cells were incubated for 16 h at37° C., washed with FACS-buffer, and target cells were labeled withanti-murine antibodies; incubation was performed at RT for 30 min. Deadcells were excluded by staining with propidium iodide, and cells wereanalyzed with a FACSCalibur (Becton Dickinson). Dose-dependentcytotoxicity was observed for AchR-Fc.

EXAMPLE 24 De-Immunization of the Extracellular Domain of Human AchR-Fc

Peptide analogs to pathogenic epitopes of the human AchR alpha subunithave been described (Zisman 1996, Proc Natl Acad Sci USA 93, 4492-7). Inthis study, a single substitution (Alanine at position 207) in thepeptide encompassing region aa 195-212 was performed. This substitutedpeptide was shown to bind to human MHC molecules as efficiently as thewildtype peptide. This mutated variant could specifically inhibitstimulation of peripheral blood lymphocytes (PBLs) from MG patientsinduced by the pathogenic wildtype peptide.

The codon encoding Methionine at amino acid 207 of the AchRextracellular domain (Zisman, 1996, Proc Natl Acad Sci USA 93, 4492-7)was substituted to Ala-encoding nucleotides in an AchR-Fc cDNA viasite-directed mutagenesis by standard protocol. Briefly, primers weresynthesized covering the 5′ and 3′ terminal sequence (5′: A, 3′: D) ofthe region encoding the AchR-Fc fusion protein. For mutagenesis, primerswere chosen to include the desired nucleotide exchanges flanked by 15 to20 bases to both the 3′ and 5′ end (forward primer (5′): C, backwardprimer (3′): B). Primers A and B were used in PCR to generate fragmentI, primers C and D generated fragment II, each using wild-type AchR-Fcas a template. Following gel extraction, fragments I and II were used astemplates to synthesize mutated AchR-Fc DNA with primers A and D.

For transfection DHFR-deficient CHO cells were plated at 3×10⁵/well intissue culture 6-well plates and incubated at 37° C. overnight. 3 μg ofAchR-Fc DNA were pipetted in sterile Eppendorf tubes, supplemented with100 μl MEM-α medium (Gibco BRL) and 10 [2l SuperFect transfectionreagent (Qiagen), and incubated for 10 min at RT. 600 μl of MEM-α mediumwere added, and the reaction mixture was transferred to CHO cells. Afterincubation for 2 h at 37° C., the supernatant was aspirated, cells werewashed once with PBS, and 2 ml MEM-α medium (10% FCS, HT-supplement1:100) were added to each well. Transfection efficiency was determinedto be 10% via standard β-galactosidase control transfection. After 24 hat 37° C., transfected cells were transferred to 10 ml cell culturebottles (Nunclone Δ, Nalge Nunc International) and selected forexpression of the DHFR gene via growth in non-supplemented MEM-α mediumplus 10% dialysed FCS. Following two passages, transfectants werefurther selected by addition of 20 nM methotrexate (MTX) to theselection medium. Cells were passaged 3 times, whereafter MTXconcentration was increased to 100 nM and after further 3 passages, MTXwas added to a final concentration of 500 nM. Stably transfectedCHO-cells were transferred to 500 ml roller-bottles (Nalge NuncInternational) in MEM-α, 500 nM MTX and 2.5% dialysed FCS. Supernatantwas harvested, and recombinant protein was purified using a one-steppurification procedure via Protein A affinity chromatography (HiTrapProtein A column, Pharmacia). Protein was eluted with 20 mM citrate, pH3, using a linear gradient.

Investigation of Cytotoxic Activity

The cytotoxic potential of purified AchR-Fc fusion protein was assayedusing an in-vitro test system. Briefly, anti-AchR mouse hybridoma cellswere exposed to serum-free medium (Hybridoma SFM, Gibco) and selectedfor expression of cell surface-bound immunoglobulin. The establishedanti-AchR positive hybridoma cell line was used as a target cell line ina FACS-based in-vitro cytotoxicity assay. The anti-AchR hybridoma cellline was adapted to serum-free medium (Hybridoma SFM, Gibco). Cells werepassaged 1:5 every third day, and cultured in 100% SFM for a period of4-5 months. Thereafter, AchR-reactivity was analyzed by FACS analysisfor surface binding of biotinylated AchR protein. Cells showed cellsurface expression of murine immunoglobulin and bound to recombinantAchR. The FACS-based assay was performed using freshly isolated humanPBMCs as effector cells (ECs). ECs and target cells were incubatedovernight at 37° C./5% CO₂ at an E:T ratio of 10:1 and serial dilutionsof fusion protein added in a constant volume of 20 μl to 180 μl cellsuspension. Cells were stained with FITC-labeled goat-anti-mouse Igantibody and propidium iodide. The live target cell population wasmeasured as percentage of the whole cell population analyzed. Unspecificbackground was measured in the absence of protein. Cytotoxicity (CT) wascalculated as CT=100×(1-(live target cells in sample/live target cellsin control)) and background staining of human PBMCs incubated alone wassubtracted. The AchR-Fc protein was found to specifically depleteAchR-reactive hybridoma cells in the presence of PBMCs.

Investigation of T-Cell Stimulation with Mutated AchR-Fc Protein

Proliferation of PBLs in response to stimulation with wildtype andde-immunized AchR-Fc proteins was investigated. PBLs of MG patients werecollected by Ficoll density gradient centrifugation. Cells were culturedin 96-well microtiter plates with various concentrations of proteins.After incubation for 6 days, cells were pulsed with 3-H thymidine. 16hlater, cells were harvested on a filter paper and radioactivity wasassessed in counts per minute (cpm). PBLs stimulated with thedeimmunized AchR-Fc protein showed a significantly lower proliferativeresponse compared to the wildtype AchR-Fc protein.

Induction of EAMG and Investigation of B-cell Modulation by RecombinantAchR-Fc Protein

For induction of EAMG and clinical evaluation, female Lewis rats, 6-7weeks of age, were injected once in the hind foot pads with 40 μg ofAchR purified from the electric organ of Torpedo californica (Aharonov,1977, Immunochemistry 14, 129-137) emulsified in complete Freund'sadjuvant containing 1 mg/rat Mycobacterium tuberculosis (Difco). EAMGwas evaluated as follows: grade 0, no weakness or fatigability; grade 1,weak grip and fatigability; grade 2, weakness, hunched posture at rest,decreased body weight, tremolousness; grade 3, severe weakness, markeddecrease in body weight, moribund; grade 4: dead. Animals were weighedand evaluated weekly up to 7-9 weeks after immunization with TorpedoAchR.

Treatment with AchR-Fc fusion proteins was started 15 days before, 6days before, 3 days after, or 7 days after immunization with TorpedoAchR. AchR-specific antibodies were assayed by ELISA as described(Barchan 1998, Eur. J. Immunol. 28, 616-624). Bound antibodies weredetected by alkaline phosphatase-conjugated goat anti-rat IgG followedby measuring the enzymatic activity of alkaline phosphatase. Results areexpressed as OD at 405 nm. Antibody titers against AchR weresignificantly reduced in those groups of animals treated with therecombinant AchR-Fc protein suggesting a depletion of autoreactive Bcells directed against AchR.

EXAMPLE 25 General Principle for Deimmunization of Autoantigens Linkedto an Effector Domain

Fusion proteins of autoantigens and an effector domain, p.e. theimmunoglobulin Fc part or anti CD3 immunoglobulin part were constructedand purified according to the methods applied for MOG-Fc and MOG×CD3.Specific binding of these autoantigen fusion proteins to B-cells wasverified using hybridoma cells selected for expression of cell-surfaceautoantigen-specific immunoglobulin. Specific binding of the effectordomain was analyzed using T cells or Fc receptor bearing cells.Depletion of autoreactive B-cells induced by these fusion proteins wasanalyzed in a cytotoxicity assay using autoantigen-reactive hybridomacells.

In order to remove immunodominant T-cell epitopes from the autoantigenfusion proteins without affecting their capacity to eliminateautoreactive B-cells identification of T cell epitopes was performed asfirst step. Identification of T-cell epitopes could be performed by a)Peptide Threading which is based on the analysis of peptides that bindto MHC class II molecules (as described by Biovation): By combiningknown HLA three dimensional structures and homology modelling, thestructures of many human MHC alleles could be predicted. Overlappingpeptides from the autoantigen protein sequence were assessed for bindingto MHC classeII in silico and a binding score was calculated; b)Peptide-MHC binding in vitro. Peptide-MHC binding was analyzed using acollection of human cell lines carrying a repertoire of different MHCclass II alleles. Synthetic peptides from antibody and protein sequenceswere tested for displacement of control biotinylated peptides. Followingcell lysis, MHC class II molecules were immunoprecipitated and testedfor peptide binding using avidin-enzyme conjugates; c) Human T cellassays measuring the T cell response to peptides presented inconjunction with MHC class II molecules: Proteins were mixed with cellfractions containing human antigen presenting cells and T cell fractionswere added. T cell proliferation in response to the specific antigens isthen assessed by 3-H thymidine uptake or cytokine measurement. Human Tcell assays could be used to identify peptide-MHC class II complexeswhich can trigger T cell responses; d) Alanin substitution of singleamino acids in overlapping peptides corresponding to the autoantigen:The Ala-substituted peptides are tested for their capacity to induceT-cell proliferation in a 3-H thymidine uptake assay; e) class IItetramer epitope mapping (Kwok, 2001, Trends in Immunology 22, 583-588;f) searching a MH C-binding motif database (p.e. www.wehil.wehi.edu.au,www.syfpeithi.bmi-heidelberg.com/Scripts/MHCServer.dII/Info.htm,www.cancerimmunity.org/peptidedatabases/Tcellepitopes.htm) or publisheddata concerning mapping of T cell epitopes of a certain autoantigen; g)ELISPOT assay or h) cytokine pattern analysis on mRNA level.

The potential MHC class 11 binding motifs identified by the use of themethods described above could be eliminated from the autoantigenmolecule by substitution of a single or more amino acids within the MHCclass II binding peptide preferably to alanine. Such substitutions willeliminate or greatly reduce binding to MHC class II, Alternatively, MHCbinding peptide could be altered to a sequence which retains its abilityto bind MHC class II but fails to trigger T cell activation.Modifications may also comprise deletion of one or more amino acids ofthe epitope. Such modifications can be introduced into the peptide bystandard chemical peptide synthesis.

Deimmunized fusion proteins consisting of an autoantigen and an effectordomain were tested for their capacity to eliminate autoreactive B-cells.Depletion of autoreactive B-cells could be tested by a) an in vitrocytotoxicity assay with hybridoma cells expressing autoantigen-specificantibodies on the cell surface. This cytotoxicity assay could beperformed as FACS-based assay or as 51-Cr release assay. Freshlyisolated human PBMCs were used as effector cells. b) animal models basedon immunization with autoantigens. Animals developed disease and hightiters of autoantigen-specific antibodies reflecting the presence ofautoreactive B-cells. Treatment with deimmunized fusion proteins of anautoantigen and an effector domain induced a decrease of antibody titerssuggesting a depletion of autoreactive B-cells. Antibody titers could bedetected with ELISA, EIA, radioimmunoassay, c) a transgenic mouse modelexpressing immunoglobulin specific for the autoantigen. This model didnot require immunization with the autoantigen but animals could bedirectly treated with deimmunized fusion proteins of an autoantigen andan effector domain. The number of autoreactive B-cells could be detectedby FACS analysis, autoreactive antibody titers could be detected byELISA.

Effect of deimmunized fusion proteins of an autoantigen and an effectordomain on T-cell activation was determined by a) T-cell stimulationassay using an autoantigen-reactive T-cell line: An autoantigen-reactiveT-cell line was prepared by immunization of transgenic mice (human MHCclass II or human MHC class II/human TCR) with the recombinantautoantigenic protein. 8 days following immunization, spleen anddraining lymph nodes were prepared and single-cell cultures wereestablished. Cells were re-stimulated with irradiated antigen-presentingcells (APC) loaded with the deimmunized autoantigen, thereby selectingfor autoantigen-reactive T cells. This T-cell line was used in a T-cellproliferation assay and the proliferative response of theautoantigen-reactive T-cell line was tested in a standard 3-H thymidineincorporation assay; b) measuring T-cell stimulation with thedeimmunized fusion protein of an autoantigen and an effector domain.Proliferation of human PBLs derived from a patient suffering from anautoimmune disease in response to stimulation with deimmunized andnon-deimmunized fusion protein was investigated. PBLs were collected byFicoll density gradient centrifugation and cultured in 96-wellmicrotiter plates with various concentrations of proteins. After 6 days,cells were pulsed with 3-H thymidine. 16 h later, cells were harvestedon a filter paper and radioactivity was assessed in counts per minute(cpm). PBLs stimulated with the deimmunized fusion protein showed asignificantly lower proliferative response as compared to thenon-deimmunized fusion protein; c) assaying cytokine patterns of primarymurine T cells on protein level or on mRNA level (RT-PCR): Transgenicmice (human MHC class II or human MHC class II/human TCR) were immunizedwith deimmunized fusion protein of an autoantigen and an effector domainand spleen and draining lymph nodes were prepared and blood was taken.Single-cell cultures were established and the Th (T-helper cell)cytokine profile in the supernatant was detected by ELISA. In contrastto mice immunized with the non-deimmunized fusion protein, miceimmunized with the deimmunized molecule did not display a strong Th1cytokine profile (IFNγ high, TNFαhigh); d) immunization of transgenicmice (human MHC class II or human MHC class II/human TCR) with thedeimmunized fusion protein of an autoantigen and an effector domain anddetermination of the absence of disease induction. In contrast to thedeimmunized fusion protein, immunization with the non-deimmunized fusionprotein led to a rapidly progressive disease; e) immunization ofprimates with the deimmunized fusion protein of ah autoantigen and aneffector domain and determination of the absence of disease induction.In contrast to the deimmunized fusion protein, immunization with thenon-deimmunized fusion protein led to a rapidly progressive disease orf) ELISPOT assay.

1. A (poly)peptide construct consisting of at least two domains or of atleast two pluralities of domains wherein one of said domains orpluralities of domains comprises a de-immunized, autoreactive antigen or(a) fragment(s) thereof specifically recognized by the lg receptors ofautoreactive B-cells and wherein a/the further domain or plurality ofdomains comprises an effector molecule capable of interacting withand/or capable of activating NK-cells, T-cells, macrophages, monocytesand/or granulocytes and/or capable of activating the complement system.2. The (poly)peptide construct of claim 1, wherein said construct is afusion (poly)peptide or a mosaic (poly)peptide.
 3. The (poly)peptideconstruct of claim 1, wherein said construct is a cross-linked(poly)peptide construct.
 4. The (poly)peptide construct of claim 1,wherein said autoreactive antigen or (a) fragment(s) thereof is selectedfrom the group consisting of intracellular matrix proteins,extracellular matrix proteins, complement factors, nuclear antigens,cell surface receptors, nuclear receptors, lipoproteins, solublefactors, membrane proteins, heat shock proteins, proteins with sequencesimilarity to microbial antigens or to dietary components and proteinsof intercellular structures.
 5. The (poly)peptide construct of claim 4,wherein said intracellular matrix protein is selected from the groupconsisting of keratin, filaggrin, antiperinuclear factor
 7. 6. The(poly)peptide construct of claim 4, wherein said extracellular matrixprotein is collagen, wherein said complement factor is C5, wherein saidnuclear antigen is selected from the group consisting of histones,snRNPs, topoisomerase I, ro (SS-A-Ro), Ia (SS-B-La), ScI-70, centromerprotein (CENP), A2, Sm proteins, tRNA synthetase and Ku antigen, whereinsaid cell surface receptor is selected from the group consisting ofTSH-receptor, Ach-receptor, asialo-glycoprotein receptor and plateletintegrin GpIIb:IIIa, wherein said soluble factor is selected from thegroup consisting of i-antigen, Rh-blood group factor, 21-hydrolaseenzyme, glutamic acid decarboxylase (GAD), insulin, (ICA) 512, ICAP-69,(tissue) transglutaminase (tTG), transaldolase, S100beta, oxidizedlow-density lipoprotein (ox-LDL), CNPase, proteinase 3 and type Iantigen, wherein said membrane protein is selected from the groupconsisting of PLP, MAG, MBP, MOG, Golgi-proteins, cytochrome P450(CYPs), UDP-glucuronosyltransferase (UGTs), pemphaxin, LAD285, type XVIIcollagen, wherein said heat shock protein is selected from the groupconsisting of alpha B-crystallin, Hsp27, HSP70 and HSP60, wherein saidproteins with sequence similarity to microbial antigens or to dietarycomponents are selected from the group consisting of antigens mimickingproteins, polypeptides and/or carbohydrate structures fromStreptococcus, Klebsiella, Proteus, M. tuberculosis, adenovirus,poliovirus, retrovirus, papillomavirus, measles virus, gluten orbutyrophilin, and wherein said protein of intercellular structures isselected from the group consisting of desmoglein-1 (Dsg1), desmoglein-3(Dsg3), desmocollin, desmoplakin, envoplakin, periplakin, BPAG-1(BP230), BPAG-2 (BP180) and/or HD1/plektin.
 7. The (poly)peptideconstruct of claim 1, wherein said T cells are cytotoxic T cells.
 8. The(poly)peptide construct of claim 1, wherein said effector molecule is areceptor-ligand or the Fc-part of an immunoglobulin.
 9. The(poly)peptide construct of claim 8, wherein said effector moleculespecifically binds to a molecule of the CD3-receptor complex.
 10. The(poly)peptide construct of claim 8, wherein said receptor-ligand is anantibody or (a) fragment(s) or derivative thereof or an aptamer.
 11. The(poly)peptide construct of claim 10, wherein said antibody derivative isa scFv directed against a molecule of the CD3 receptor complex.
 12. The(poly)peptide construct of claim 1, wherein said domain comprising ade-immunized, autoreactive antigen or (a) fragment thereof isde-immunized MOG (eMOG) or (a) fragment(s) thereof and wherein a/theother domain comprising an immunological effector molecule is ananti-CD3 receptor or an Fc-part of an immunoglobulin.
 13. The(poly)peptide construct of claim 12, wherein said (poly) peptideconstruct is encoded by (a) a polynucleotide comprising a nucleic acidmolecule encoding the polypeptide as depicted in SEQ ID NO. 28; (b) apolynucleotide comprising a nucleic acid molecule having the DNAsequence as depicted in SEQ ID NO. 27; (c) a nucleotide sequencehybridizing to a sequence which is complementary to a nucleotidesequence of (a) or (b); or (d) a nucleotide sequence being degenerate tothe sequence of the nucleotide sequence of (c).
 14. The (poly)peptideconstruct of claim 13, wherein said (poly)peptide construct is a(poly)peptide comprising the amino acid sequence as depicted in SEQ IDNO.
 28. 15. A (poly)nucleotide encoding at least one (poly)peptideconstruct as defined in claim
 1. 16. A vector comprising a(poly)nucleotide as defined in claim
 15. 17. The vector of claim 16,wherein said vector is an expression vector.
 18. A host transformed withthe vector of claim
 16. 19. The host of claim 18, wherein said host is amammalian cell.
 20. A composition comprising: 1) the polypeptideconstruct of claim 1, 2) a polynucleotide encoding at least onepolypeptide construct as defined in claim 1, 3) a vector comprising apolynucleotide encoding at least one polypeptide construct as defined inclaim 1, or 4) a host transferred with a vector comprising apolynucleotide encoding at least one polypeptide construct as defined inclaim
 1. 21. The composition of claim 20 for the selective eliminationof autoreactive B-cells.
 22. The composition of claim 20 furthercomprising a compound capable of selectively eliminating plasma cellsand/or a compound capable of selectively eliminating (an)auto-antibody(ies).
 23. The composition of claim 22, wherein saidcompound capable of selectively eliminating plasma cells is an antibodyor (a) fragment(s) or a derivative thereof specifically detecting anplasma cell-specific epitope.
 24. The composition of claim 22, when saidcompound capable of selectively eliminating (an) auto-antibody(ies) isan anti-idiotypic antibody or (a) fragment(s) or a derivative thereofspecifically reacting with said auto-antibody(ies).
 25. The compositionof claim 20 which is a pharmaceutical composition optionally comprisinga pharmaceutically acceptable carrier.
 26. The composition of claim 20for selective reduction of autoreactive immunoglobulins. 27-29.(canceled)
 30. A method of therapy, amelioration and/or prevention of anautoimmune disease comprising the administration to a subject in need ofsuch therapy and/or prevention an effective amount of at: 1) at leastone polypeptide construct of claim 1, 2) at least one polynucleotideencoding at least one polypeptide construct as defined in claim 1, 3) atleast one vector comprising a polynucleotide encoding at least onepolypeptide construct as defined in claim 1, or 4) at least one hosttransferred with a vector comprising a polynucleotide encoding at leastone polypeptide construct as defined in claim
 1. 31. The method of claim30, wherein said autoimmune disease is selected from the groupconsisting of Pemphigus vulgaris, Bullous pemphigoid, Goodpasture'ssyndrome, autoimmune haemolytic anemia (AIHA), rheumatoid arthritis,Systemic Lupus erythematosus, Grave's disease (autoimmunehyperthyroidism), contact dermatitis, Myasthenia gravis, juvenilediabetes, Sjogren's syndrome, autoimmune thyroiditis, primaryhypoadrenalism (Addison's disease), multiple sclerosis thrombocytopenicpurpura, Morbus Wegener (granulomatosis), pemphigous foliaceous, linearIgA dermatosis and celiac disease.